STUDIES  ON  GIABDIA  MICROTI 


A  THESIS  SUBMITTED  IN  PARTIAL  SATISFACTION  OF 

THE  EEQUIEEMENTS  FOR  THE  DEGREE  OF 

DOCTOR  OF  PHILOSOPHY 

AT  THE  UNIVERSITY  OF  CALIFORNIA 


BY 


WILLIAM  CHARLES  BOECK 


1918 


UNIVERSITY  OF  CALIFORNIA  PUBLICATIONS 

IN 

ZOOLOGY 

Vol.  19,  No.  3,  pp.  85-134,  plate  1,  19  figures  in  text  April  7,  1919 


STUDIES  ON  GIARDIA  MICROTI 


BY 
WILLIAM  C.  BOECK 


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from  the   Sierra  Nevada  and   Sjrstematic  Notes   on   Other   California 
Lizards,  by  Charles  Lewis  Camp.    Pp.  63-74.    December,  1916 .10 


UNIVERSITY  OF  CALIFORNIA   PUBLICATIONS 

IN 

ZOOLOGY 

Vol.  19,  No.  3,  pp.  85-134,  plate  1,19  figures  in  text  April  7,  1919 


STUDIES  ON  GIARDIA  MICROTI 


BY 

WILLIAM  C.  BOECK 


CONTENTS 

PAGE 

The  cycle  of  encystment  in  Giardia  microti 86 

Introduction 86 

Materials  and  Methods 87 

Discussion 104 

Summary 107 

The  development  of  Giardia  microti  within  the  cyst 108 

Materials  and  data 108 

Binary  fission  within  the  cyst 110 

Multiple  fission  within  the  cyst 113 

Summary 118 

The  parabasal  bodies  of  Giardia  microti 102 

Function  of  the  parabasal  bodies 120 

Biochemical  and  staining  qualities  of  the  parabasal  bodies  122 

Origin  of  the  parabasal  bodies 124 

Summary 125 

The  therapeutic  value  of  bismuth  subnitrate  and  bismuth  salicylate  in  the 

treatment  of  giardiasis  (lambliasis)  of  rats 126 

Introduction 126 

Procedure 126 

Discussion 130 

Summary 132 

Literature  cited 133 

Explanation  of  plate 134 


86  University  of  California  Puhlications  in  Zoology       [Vol.  19 

THE  CYCLE  OF  ENCYSTMENT  IN  GIARDIA  MICROTI 

Introduction 

Most  of  the  Protozoa  which  have  been  studied  intensively  for  a 
long  period  of  time  have  been  shown  to  possess  rhythms  or  cycles  of 
what  might  be  termed  general  vitality.  These  rhythms  may  be  seen 
in  the  sensitiveness  to  environmental  conditions  of  the  individual 
organism  or  races  of  organisms,  as  in  the  case  of  Paramoecium  aurelia 
(Woodruff),  but  more  often  the  rhythms  or  cycles  are  evident  in 
the  reproductive  activity  of  the  protozoan.  Cycles  in  the  fission  rate  of 
Paramoecium  were  found  by  Calkins  (1904)  and  by  Woodruff  (1905). 
Gregory  (1909)  showed  that  there  were  cycles  of  high  and  low  vitality 
in  Stylonychia  mytilus  and  Tillina  magna.  The  cycles  were  found  to 
be  fairly  regular  and  the  last  work  of  Woodruff  on  Paramoecium 
aurelia  (1917)  has  shown  that  even  changes  in  the  culture  media  and 
in  temperature  fail  to  modify  the  cycles  of  endomitis  which  are 
characteristic  for  the  species.  There  may  be  a  slight  initial  influence 
on  the  cycle  of  the  Paramoecium  when  the  ciliates  are  suddenly  placed 
in  a  changed  environment,  but  after  a  short  period,  during  which  a 
readjustment  of  the  organisms  takes  place,  the  endomitic  interval 
regains  its  normal  length. 

Cycles  in  the  life  history  of  Haematozoa  are  also  known,  the  most 
classical  examples  of  which  are  those  of  the  various  species  of  Plas- 
modium. In  these  protozoans  not  only  is  the  reproductive  process 
rhythmic  in  nature,  but  also  that  of  sporulation. 

In  the  flagellates  the  presence  of  cycles  of  vitality  expressed  in 
reproductive  activities  is  also  known.  The  haemoflagellates  in  their 
life  history  present  striking  examples  of  cyclic  development,  and  one 
has  only  to  watch  the  various  free  living  flagellates  in  an  ordinary 
aquarium  to  notice  that  a  rhythm  or  cycle  must  exist  for  them  in  view 
of  the  fact  that  their  numbers  are  seen  to  fluctuate  from  day  to  day 
and  week  to  week. 

For  some  time  a  cycle  of  encystment  was  suspected  in  Giardia 
found  in  the  intestine  of  rodents,  but  it  was  during  an  investigation 
of  mitosis  in  Giardia  microti  (Boeck,  1917)  that  evidence  of  such  a 
cycle  was  found.  A  table  in  the  paper  referred  to  above  showed  that 
cysts  were  found  upon  examination  in  only  five  out  of  nineteen  cases 
of  infection.  This  fact  raised  the  suspicion  of  the  presence  of  a  cycle 
of  encystment,  for  if  encystment  occurs  continuously  in  the  life  history 


1919]  Boeck:  Studies  on  Giardia  Microti  87 

of  each  flagellate,  then  cysts  should  be  present  in  every  one  of  the 
hosts  in  which  the  examination  of  the  large  intestine  had  been  made. 
(The  large  intestine  is  the  region  of  the  digestive  tract  in  which  cysts 
occur  in  greatest  numbers.)  Again,  if  encystment  occurs  at  all  times 
then  cysts  should  always  be  found  in  faeces  of  rats  infected  with 
Giardia,  and  the  number  of  cysts  in  the  faecal  sample  of  a  rat  should 
be  approximately  the  same  for  each  day.  The  process  of  encystment 
would  then  be  an  even,  regular  process  from  day  to  day  and  would 
not  show  evidence  of  a  sudden  rise  and  fall  in  the  number  of  cysts 
found  in  the  faeces. 

The  enumerations  of  the  cysts  of  G.  intestinalis  in  human  dysen- 
teric faeces  made  by  Porter  (1916)  gave  for  the  first  time  critical  evi- 
dence of  a  cycle  of  encystment  in  this  flagellate,  which  is  a  species  allied 
to  G.  nvicroti. 

The  importance  of  knowing  whether  or  not  there  is  a  cycle  of 
encystment  in  these  flagellates  cannot  be  overestimated.  It  would  be 
of  great  significance  in  the  therapy  of  dysentery  caused  by  these 
organisms  as  well  as  a  considerable  factor  to  reckon  with  in  the 
diagnosis  of  dysenteric  patients  by  the  daily  examination  of  their 
stools,  if  a  more  or  less  regular  cycle  should  be  found  to  occur.  It 
would  be  necessary  to  make  a  longer  series  of  examinations  in  order  to 
determine  whether  or  not  a  patient  were  infected  if  a  cycle  of  encyst- 
ment is  present  in  the  life  history  of  the  flagellates  causing  the  dysen- 
tery than  it  would  if  no  such  cycle  were  present.  Accordingly  daily 
examinations  of  the  faeces  of  fifteen  rats  were  begun  and  carried  on 
throughout  a  period  of  twenty-eight  days  for  the  purpose  of  securing 
if  possible  sufficient  additional  evidence  to  definitely  determine  the 
facts. 

Materials  and  Methods 

The  faecal  pellets  of  the  rats  vary  in  size  from  the  average,  about 
twelve  millimeters  in  length  and  six  millimeters  in  diameter,  to  about 
four  millimeters  in  length  and  two  millimeters  in  diameter.  Constipa- 
tion was  present  when  the  pellets  of  the  smallest  size  were  defaecated. 
The  small  size  of  the  pellets  could  not  be  correlated  with  the  absence 
or  presence  of  cysts  within  them.  At  no  time  were  the  faeces  liquid 
in  consistency.  There  is  no  evidence  of  diarrhoea  in  the  rats  infected 
with  Giardia  like  that  caused  by  G.  intestinalis  in  man  and  mice.  The 
color  of  the  faeces  was  usually  a  dark  brown.  Variations  in  the  color 
from  darker  shades  of  brown  to  very  light  yellow  were  of  no  signi- 


Anaf^aa 


88  University  of  California  Publications  in  Zoology       ["^oi^- 1^ 

ficance  as  a  diagnostic  factor  in  the  detection  of  cysts.  The  amount 
of  stools  defaecated  each  day  varied  and  on  no  day  did  the  rats  fail 
to  pass  any  stools. 

In  the  examination  of  the  stools  for  the  first  four  days  a  modifi- 
cation (Boeck,  1917)  of  the  method  of  faecal  examination  described 
by  Cropper  and  Row  (1917)  was  used.  The  method  in  its  modified 
form  is  as  follows:  To  at  least  one  gram  of  faeces  add  thirty  cubic 
centimeters  of  normal  salt  solution  and  stir  with  a  Hamilton-Beach 
** cyclone"  mixer  for  ten  minutes.  Then  add  five  cubic  centimeters  of 
ether  and  stir  for  two  minutes  longer.  The  suspension  is  then  placed 
in  a  separatory  funnel  and  allowed  to  stand  for  five  to  seven  minutes, 
during  which  time  the  two  liquids  will  separate,  the  ether  carrying 
most  of  the  debris  to  the  top  while  the  cysts  remain  in  the  normal 
salt  solution  below.  The  normal  salt  solution  is  then  drawn  off  into 
a  centrifuge  tube  of  a  capacity  of  fifteen  cubic  centimeters  and  centri- 
fuged  for  three  minutes.  The  cysts  are  concentrated  at  the  bottom 
of  the  tube  and  the  supernatant  fluid  is  drawn  off  with  a  pipette. 
A  drop  of  neutral  red  solution,  one  part  to  ten  thousand  parts  of  dis- 
tilled water,  is  added  to  a  drop  of  the  residue  from  the  bottom  of  the 
tube  and  transferred  to  a  slide  for  microscopic  examination.  The 
cysts  are  readily  detected  with  a  one-inch  eye-piece  and  a  four  milli- 
meter objective.  The  cysts  measure  about  fourteen  microns  in  length 
and  six  to  seven  microns  in  diameter. 

The  other  examinations  were  made  by  making  a  suspension  of  the 
stools  in  distilled  water,  and  stirring  them  until  the  mixture  appeared 
uniform  in  density.  Again  a  drop  of  neutral  red  solution  of  the  same 
dilution  as  in  the  previous  method,  was  added  to  a  sample  of  the  faecal 
suspension,  which  was  then  examined  under  the  microscope  for  cysts. 
The  neutral  red  is  of  great  service  in  that  it  differentiates  the  cyst 
from  the  yeasts  and  debris,  which  in  most  cases  are  partially  or  totally 
stained  while  the  cysts  are  very  seldom  affected  by  the  stain  and  so 
stand  out  in  the  preparation  as  clear,  transparent,  ovoid  bodies.  The 
cysts  also  reveal  in  many  instances  their  two  or  more  nuclei,  also  the 
remains  of  the  axostyle,  intracytoplasmic  flagella,  and  the  parabasal 
bodies. 

There  is  no  doubt  that  the  concentration  of  the  cysts  by  the  ether- 
centrifuge  method  is  superior  in  accuracy  to  the  simple  microscopical 
examination  described  in  the  preceding  paragraph,  but  because  of  the 
number  of  rats  under  observation  this  shorter  method  was  used  in  this 
work.  A  count  of  the  cysts  was  also  undertaken  at  the  time  examina- 
tions were  made.     At  first  a  haemocytometer  was  used,  but  it  was 


1919]  Boeck:  Studies  on  Giardia  Microti  89 

found  that  the  cysts  were  never  in  sufficient  numbers  to  make  the  use 
of  this  instrument  practicable.  The  number  of  cysts  were  then  counted 
in  any  twenty  fields  of  the  microscope,  using  a  one-inch  ocular  and 
four-millimeter  objective.  The  following  table  (1)  shows  the  occur- 
rence of  the  cysts  in  the  faeces  and  the  count  that  was  made.  The 
examinations  commenced  October  25, 1917,  and  were  concluded  Novem- 
ber 21,  1917. 

This  table  is  a  record  of  the  daily  examination  of  the  faeces  of 
the  different  rats,  with  the  number  of  cysts  counted  in  any  twenty 
fields  of  the  microscope.  A  negative  sign  signified  that  no  cysts  were 
found  in  the  faeces  for  that  day. 

From  a  study  of  table  1  it  will  be  seen  that  rat  number  7  was  nega- 
tive for  all  the  examinations.  It  received  five  treatments  of  magnesium 
sulphate  in  a  twenty-five  per  cent  solution  and  no  cysts  or  living 
Giardia  were  seen  in  the  semi-fluid  stools.  It  was  concluded  that  this 
rat  was  not  infected  with  Giardia.  Cysts  were  found  in  the  faeces  of 
rat  3  for  three  successive  days,  after  which  there  was  no  recurrence. 
In  rat  15  cysts  appeared  November  8  and  not  again  until  November 
20  and  21,  when  the  examinations  for  all  the  rats  were  concluded. 

In  the  study  of  the  rats  in  table  1,  to  determine  whether  or  not  there 
w^as  evidence  for  a  cycle  of  encystment,  the  data  for  rats  3  and  15  were 
not  considered  since  neither  showed  two  complete  periods  when  cysts 
w^ere  ejected  and  consequently  no  interval  could  be  determined  between 
successive  appearances  of  cysts.  The  data  for  rats  1,  2,  4,  5,  6,  8,  9,  10, 
11,  12,  13,  and  14  are  the  only  data,  then,  that  were  used  in  this  study 
of  periodicity  in  the  appearance  of  the  cysts. 

The  length  of  the  period  during  which  the  cysts  were  defaecated 
with  the  faeces  varied  from  one  to  fourteen  days,  and  the  interval 
during  which  no  cysts  were  found  in  the  faeces  varied  from  one  to 
eleven  days.  There  is  a  common  feature  seen  in  the  records  of  most 
of  the  rats  in  that  the  ejection  of  cysts  occurred  at  three  or  four 
periods  during  the  twenty-eight  days  of  daily  examinations.  The 
highest  count  of  cysts  was  recorded  on  November  21  in  the  examina- 
tion of  the  faeces  of  rat  14.  There  were  eighteen  cysts  in  twenty 
fields  of  the  high  power  objective. 

The  data  for  each  one  of  the  rats  was  plotted  so  that  each  graph 
resulting  would  represent  more  clearly  the  evidence  for  the  presence 
of  a  cycle  of  encystment  in  Giardia  microti.  The  points  on  the  ab- 
scissa represent  the  days  when  the  examinations  were  made,  while  the 
points  on  the  ordinate  represent  the  number  of  cysts  counted  in  twenty 


90  University  of  California  Publications  in  Zoology        [Vol.  19 


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1919] 


Boeck:  Studies  on  Giardia  Microti 


91 


fields  of  the  high  power  objective.    Each  one  of  these  graphs  will  now 
be  discussed. 

Kat  1.  This  graph  is  plotted  from  the  data  of  rat  1.  Its  modes  are 
very  sharply  defined  since  the  periods  of  depression  are  characterized 
by  an  absence  of  cysts.  The  first  period  when  cysts  were  found  in  the- 
faeces  lasted  from  October  29  to  November  1.  The  mode  of  this  part 
of  the  curve  was  reached  on  October  30.  A  depression  period  of  two 
days  when  no  cysts  were  found  in  the  faeces  ensued.  This  period 
endured  for  three  days.  The  mode  recording  the  maximal  number  of 
cysts  occurred  on  November  5.    A  period  when  no  cysts  were  found 


1  2  3  4   5   6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28 

Eat  1 
Fig.  1 

in  the  faeces  followed  for  four  days,  then  there  were  two  days  when 
one  cyst  was  detected,  followed  again  by  an  examination  in  which  no 
cysts  were  detected.  Another  period  of  cysts  in  the  faeces  took  place 
from  November  12  up  to  November  21,  when  the  last  examination  was 
made.  The  mode  of  this  period  came  on  November  15,  while  another 
mode  was  in  the  process  of  formation  because  the  number  of  cysts 
was  increasing  when  the  last  examination  was  made.  The  interval 
between  mode  one  and  two- is  six  days,  and  the  second  and  third 
modes  are  separated  by  an  interval  of  ten  days. 

Rat  2.  This  graph,  made  from  the  data  of  rat  2,  is  very  similar 
to  the  graph  in  figure  1,  there  being  three  modes  in  the  curve.  The 
first  mode  occurred  on  October  31  when  four  cysts  were  counted  in 
twenty  fields  and  the  period  of  depression  followed,  during  which  the 
number  of  cysts  decreased  to  two  on  November  2.  The  cysts  still 
continued  to  be  found  in  the  faeces  and  their  number  began  to  increase 
until  another  mode  was  reached  on  November  5.    A  sharp  falling  off 


92 


University  of  California  Publications  in  Zoology       \yoi..  19 


in  the  number  of  cysts  then  took  place,  but  the  cysts  were  not  absent 
from  the  stools  until  November  12.  Two  cysts  were  found  on  the  fol- 
lowing day,  but  none  occurred  on  November  14.  The  third  period  of 
cysts  in  the  faeces  began  November  15,  and  although  there  was  a 
decrease  in  the  number  of  cysts  on  November  18,  to  rise  again  on  the 
following  day,  this  period  may  best  be  regarded  as  continuing  from 


25  26  27  28  29  30  31  1  2  3  4   5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21 

Eat  2 
Fig.  2 


25  26  27  28  29  30  31  1  2   3  4 


10  11  12  13  14  15  16  17  18  19  20  21 


6  7  8 

Eat  3 
Fig.  3 

November  15  to  21,  when  no  cysts  were  found  in  the  last  faecal  exam- 
ination. The  intervals  between  modes  one  and  two,  two  and  three, 
are  five  and  eleven  days,  respectively. 

Rat  3.  There  was  only  one  period  when  cysts  were  ejected,  Novem- 
ber 5  to  7,  and  because  they  never  recurred  again  the  data  from  this 
rat  could  not  be  used  to  contribute  any  evidence  on  our  problem. 
The  rat  was  found  free  from  infection  at  autopsy  on  January  9,  1918 ; 
the  organs  were  normal,  and  none  of  the  lesions  of  the  intestine  were 
present  which  characterize  an  infection  by  Giardia.      The  fact  that 


1919] 


Boeck:  Studies  on  Giardia  Microti 


93 


this  rat  was  infected  and  then  became  free,  showed  that  it  was  capable 
of  throwing  off  the  infection.  There  was  no  reinfection  as  can  be  seen 
from  the  records,  which  run  negative  for  thirteen  days. 

Eat  4.  The  degree  of  infection  was  very  light  in  this  animal  for 
the  greatest  number  of  cysts  found  in  the  faeces  was  four  on  November 
10  and  11.     There  were  two  periods  when  cysts  were  found  in  the 


10 


— ^ — th rrT"T: ""n x " 

1                                                                         1 

.__ij — J — 1— ^p_i \ ^ — j__ 

:4t!:+:     _.=[    -::i . j-j.-    ' -r      +             """q4:      :-l:_  -  Ji-I-"'''" 

-!±:iq44:iiz"-:-:=:==:::::::ii4::::i"::i::-:^i:^    H: 

:4r:i4t::±::±i::ii  ~ipi:::::::  i^vit;- -:._:::rq-l:d±: 

:-^zzz^zzz:tzzzzzz-zzzzzzzzzzzzhi^zzzzzzz--ri^zz 

ttm±H 

:-— * ~rT"/-S- f:——^-^^-±rt^t-^- 

25  26  27  28  29  30  31  1  2   3  4   5   6  7  8  9  10  U  12  13  14  15  16  17  18  19  20  21 

Eat  4 
Fig.  4 


^; 


i 


25  26  27  28  29  30  31  1  2   3  4   5   6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21 

Eat  5 
Fig.  5 

faeces.  The  first  period  was  two  days  in  length  and  only  one  cyst 
was  found  on  each  day.  Then  came  an  interim  of  depression  of  six 
days  in  which  the  examinations  revealed  no  cysts.  The  mode  of  the 
second  period  of  cysts  in  the  faeces  came  on  November  10,  and  the 
period  ended  November  19.  The  interval  between  the  two  modes  is 
seven  days. 

Rat  5.  The  first  period  of  cysts  in  the  faeces,  October  30  to  Novem- 
ber 1,  results  in  a  portion  of  the  graph  sharply  set  aside  from  the 


94 


University  of  California  Puhlications  in  Zoology       ["^ol.  19 


remainder,  for  after  this  period  there  follows  an  interval  of  depression 
when  eight  negative  examinations  occurred.  The  next  period  of  cysts 
in  the  faeces  came  on  November  5,  when  the  number  of  cysts  increased 
very  suddenly  to  six,  then  fell  to  one  on  the  next  day,  and  remained 
at  two  or  one  for  the  five  following  days.  The  mode  of  this  period 
came  on  the  first  day,  when  six  cysts  in  twenty  fields  were  counted. 
No  cysts  were  counted  on  November  15,  but  on  the  next  day  the  num- 
ber of  cysts  in  the  faeces  began  steadily  to  increase  until  the  mode  of 
this  period  was  reached  on  November  20,  when  ten  cysts  were  counted. 
The  number  began  to  decrease  when  the  last  examination  was  made. 
The  intervals  between  modes  one  and  two,  two  and  three,  are  eight  and 
twelve  days,  respectively. 


25  26  27  28  29  30  31  1  2   3   4   5 


10  11  12  13  14  15  16  17  18  19  20  21 


7      5 

Eat  6 
Fig.  6 

Rat  6.  This  graph  shows  that  two  periods  of  cysts  in  the  faeces 
occurred.  These  two  periods  were  recorded  in  the  first  eleven  exam- 
inations; the  remaining  examinations  did  not  reveal  any  cysts.  The 
first  period  lasted  only  one  day,  while  the  second  period  lasted  four 
days.  The  interval  between  the  modes  of  these  two  periods  was  six 
days. 

This  rat  at  autopsy  on  January  9  was  still  infected,  even  though 
it  had  eighteen  consecutive  negative  examinations.  The  organs  were 
normal  except  that  there  was  a  small  amount  of  gas  in  the  jejunum. 
In  other  respects  this  autopsy  was  identical  with  that  of  rat  3.  The 
fact  that  this  rat  was  still  infected  after  having  had  eighteen  consecu- 
tive negative  examinations  showed  that  reinfection  may  have  taken 
place  between  the  day  when  the  last  examination  was  made  and  the 
day  the  autopsy  was  made.     Reinfection  was  possible  for  the  cages 


1919] 


Boeck:  Studies  on  Giardia  Microti 


95 


10 


were  not  cleaned  of  their  faeces  each  day  after  the  last  faecal  exam- 
ination had  been  made.  The  cysts  which  might  have  caused  this 
reinfection  may  have  entered  the  cage  by  dropping  through  the  wire 
netting  with  faecal  pellets.  On  two  instances  an  infected  rat  became 
free  from  its  cage  and  ran  over  tops  of  the  others  and  it  is  very  likely 
that  a  few  of  its  pellets  could  have  dropped  into  the  cages  of  the 
other  rats. 

Rat  8.  There  are  three  periods  and  an  incomplete  fourth  when 
the  cysts  were  found  in  the  faeces.  The  mode  of  the  first  period  occur- 
red on  October  27.  The  period  of  depression  between  this  mode  and 
the  mode  of  the  next  period  when  cysts  were  in  the  faeces  is  very 
w^ell  defined  in  that  the  curve  decreases  abruptly  from  mode  one  until 


i 


i 


i 


u 


25  25  27  28  29  30  31  1  2   3   4   5   6   7   8   9  10  11  12  13  14  15  16  17  18  19  20  21 

Eat  8 
Fig.  7 


on  October  31  no  cysts  were  found  in  the  faeces,  then  the  curve  rises 
again  until  the  second  mode  is  reached  on  November  3.  The  period 
of  depression  between  the  second  and  third  mode  is  defined  by  four 
negative  examinations.  The  third  period  of  cysts  in  the  faeces  com- 
mences on  November  4  and  continues  until  November  16.  Two  maxima 
appear  in  the  portion  of  the  graph,  and  both  of  them  have  been  termed 
modes  since  there  is  a  distinct  period  of  depression  on  November  12, 
but  it  is  possible  that  we  have  in  reality  a  single  mode  in  this  period. 
The  next  period  starts  on  November  18  and  was  continuing  when  the 
last  examination  was  made ;  but  because  of  this  last  mode  five  may  not 
be  the  true  mode  of  this  period ;  instead  the  last  one  may  be  only  a 
stepping-stone  to  another  mode  which  would  have  been  reached  if  the 
examinations  had  continued  a  greater  length  of  time.  The  intervals 
between  the  modes  are  7i/2,  5%,  5,  and  4  days,  respectively. 


96 


University  of  California  Publications  in  Zoology        [Vol.  19 


Kat  9.  There  are  three  distinct  periods  when  cysts  were  found  in 
the  faeces  in  this  rat.  This  condition,  we  have  seen,  has  been  common 
to  many  of  the  curves  already  discussed.  The  first  period  of  cysts  in 
the  faeces  extended  from  October  29  to  November  4.  The  number  of 
cysts. reached  three  in  twenty  fields  at  the  mode  on  October  31.  A 
period  of  depression  then  took  place,  from  November  4  to  12,  during 
which  no  cvsts  occurred  in  the  faeces.     From  November  12  to  15  is 


Tl'h 


i 


t±^^ 


^mfk 


10 


25  26  27  28  29  30]  31  1  2   3   4'  5   6  7   8   9  10  11  12  13  14  15  16  17  18  19  20  21 

Rat  9 
Fig.  8 


t 


i 


n 


I 


i 


i 


^m 


25  26  27  28  29  30  31  1  2   3   4   5   6  7   8   9  10  11  12  13  14  15  16  17  18  19  20  21 

Rat  10 
Fig.  9 

the  next  period  of  cysts  in  the  faeces,  with  the  mode  on  November  13. 
A  period  of  depression  of  three  days  followed ;  no  cysts  were  counted 
when  the  examinations  were  made.  The  cysts  then  appeared  again 
on  November  18,  on  which  day  a  third  mode  is  reached.  But  as  in 
mode  five^  shown  in  figure  7,  mode  three  in  this  figure  may  have  proved 
to  be  a  part  of  the  rising  curve  following  it,  had  the  examinations  been 
continued  for  a  few  days  longer.  The  intervals  between  modes  one 
and  two  and  two  and  three  are  thirteen  and  five  days,  respectively. 

Rat  10.  There  are  four  modes  representing  the  days  when  the 
highest  number  of  cysts  were  counted  in  four  distinct  periods.  The 
first  period,  October  29  to  November  1,  was  three  days  in  length  and 


1919] 


Boeck:  Studies  on  Giardia  Microti 


97 


the  mode  came  on  October  31.  The  depression  period  between  the 
first  two  cycles  of  encystment  was  two  days  in  length  during  which  no 
cysts  were  found  in  the  faces,  Tl>e  next  period  of  cysts  in  the  faeces 
was  only  two  days  in  length,  November  3  to  5,  and  four  cysts  were 
counted  on  each  of  these  two  days.  No  cysts  were  then  found  for 
five  successive  days  when  on  November  10  and  11  one  cyst  only  was 
found.  A  depression  period  of  two  days  with  no  cysts  precedes  the 
last  cycle  of  encystment,  which  was  continuing  when  the  last  exam- 
ination was  made,  to  form  another  mode.  The  intervals  between 
modes  one,  two,  three,  and  four  are  3,  2,  7,  and  4  days,  respectively. 


10  11  12  13  14  15  16  17  18  19  20  21 


The  interval  between  the  last  mode  and  incomplete  mode  on  November 
21  is  six  days. 

Eat  11.  The  first  two  periods  or  cycles  of  encystment  were  of 
short  duration  and  the  number  of  cysts  was  never  high.  The  first 
cycle  took  place  from  November  1  to  3  and  two  cysts  were  counted 
on  each  day.  A  depression  period  of  five  days  followed  this  cycle 
and  no  cysts  were  found  in  the  faeces.  The  next  cycle,  from  November 
8  to  12,  although  longer  than  the  first  cycle,  contained  only  a  small 
number  of  cysts.  The  mode  of  this  cj^cle  appeared  on  November  9. 
Another  mode  is  designated  on  November  15,  which  may  be  a  true 
mode  since  a  period  of  depression  follows  it  for  two  days,  or  this 
portion  of  the  cycle  may  be  a  part  of  the  period  of  cysts  in  the  faeces 


98 


University  of  California  Publications  in  Zoology        [Vol.  19 


which  follows  the  period  of  depression  on  November  18,  when  a  pro- 
nounced mode  is  seen  in  the  curve.  After  November  18  a  short  period 
of  depression  is  characterized  by  a. falling  off  in  the  number  of  cysts 
found,  but  their  number  begins  to  increase  again,  and  had  reached 
fifteen  cysts  when  the  last  examination  was  made.  The  intervals 
between  the  modes  are  7%,  6,  and  3  days,  respectively. 


10 


i 


I 


s 


2 


S 


?■ 


25    26    27    28    29    30    31     1 


4      5      6     7      8 

Eat  12 
Fig.  11 


9  10  11  12  13  14  15  16  17  18  19  20  21 


25  26  27  28  29  30  31  1  2   3   4   5   6  7   8   9  10  11  12  13  14  15  16  17  18  19  20  21 

Eat  13 
Fig.  12 

Eat  12.  There  are  four  cycles  of  encystment  represented  in  the 
graph  which  was  made  from  the  data  of  rat  12.  The  cysts  were  found 
during  two  successive  days  in  the  first  cycle  October  27  to  29.  A 
period  of  depression  then  continued  for  seven  days  when  no  cysts 
were  found  in  the  faeces.  The  cycle  which  followed  was  only  one  day 
in  length  and  only  three  cysts  were  counted  in  the  examination  of  the 
faeces.  The  third  cycle  is  preceded  by  two  days  when  no  cysts  were 
found.  The  mode  of  the  third  cycle  was  reached  on  November  10 
when  three  cysts  were  found  in  twenty  fields.  A  couple  of  days  when 
the  number  of  cysts  decreased  to  one  in  twenty  fields  then  ensued,  to 


1919] 


Boeck:  Studies  on  Giardia  Microti 


99 


be  followed  by  a  cycle  when  the  number  of  cysts  reached  nine  for 
three  successive  days.  The  cysts  had  disappeared  from  the  faeces 
when  the  last  examination  was  made.  The  intervals  between  the 
modes  one,  two,  three,  and  four  are  8%?  5,  and  6  days,  respectively. 

Rat  13.  The  infection  of  the  rat  was  light,  for  the  number  of 
cysts  was  small  and  there  were  only  two  cycles  of  encystment  during 
the  twenty-eight  days.  The  first  period  when  the  cysts  were  in  the 
faeces  extended  from  November  1  to  5,  with  the  highest  number  of 


25  26  27  28  29  30'  31  1 


10  11  12  13  14  15  16  17  18  19  20  21 


3   4   5   6  7  8  9 

Rat  14 
Fig.  13 

cysts  coming  on  November  3  and  4;  then  for  a  day  no  cysts  were 
found.  The  next  period  of  cysts  in  the  faeces  was  from  November 
7  to  10.  In  this  period  there  was  one  day,  November  8,  when  no 
cysts  were  found;  but  since  the  number  of  cysts  found  in  the  other 
days  was  very  small  it  is  probable  cysts  were  present  on  November  8 
and  escaped  detection  because  of  their  small  numbers.  The  mode 
of  this  second  period  was  reached  on  November  9  when  five  cysts  were 
counted  in  twenty  fields.  The  interval  between  the  two  modes  is  six 
days  in  each  case. 

Rat  14.  The  infection  in  this  rat  was  the  heaviest  for  all  the  rats 
examined.    The  maximum  numbers  of  cysts  detected  ran  higher  than 


100 


University  of  California  Publications  in  Zoology        [Vol.  19 


the  numbers  of  the  other  rats.  There  were  three  distinct  periods  when 
the  cysts  were  found  in  the  faeces.  The  first  period  lasted  four  days, 
October  27  to  31.  The  mode  of  this  period  came  on  October  28,  when 
12  cysts  were  counted.  A  period  of  depression  of  eight  days  when 
no  cysts  were  found  in  the  faeces  preceded  the  next  period  when  the 
cysts  recurred  in  the  faeces.  This  period  was  short,  lasting  only  two 
days,  with  the  mode  on  November  7.  Two  days  followed  during  which 
the  cysts  disappeared  from  the  faeces.  Then  the  third  period  when 
cysts  recurred  again  in  the  faeces  began  November  12.  This  period 
extended  to  November  18,  when  a  period  of  depression  marked  by  a 
fall  in  the  number  of  cysts  took  place.     The  mode  was  reached  on 


10 


t 


iii 


25   26    27    28    29    30    31     1     2      3 


10    11    12    13    14    15    16    17    18    19    20  21 


5      6     7      8      9 

Eat  15 
Fig.  14 

November  16,  when  10  cysts  were  recorded  for  twenty  fields.  After 
the  single  day  when  the  number  of  cysts  decreased  to  five  the  number 
immediately  increased  and  when  the  last  examination  was  made  the 
number  had  reached  eighteen  in  twenty  fields.  This  was  the  largest 
number  of  cysts  found  in  twenty  fields  for  all  the  rats  examined.  The 
intervals  between  the  modes  one  and  two  and  two  and  three,  are  10 
and  91/2  days,  respectively. 

Rat  15.  The  infection  in  this  rat  was  very  light,  for  during  the 
twenty-eight  days  cysts  occurred  only  three  times  and  in  small  num- 
bers, though  the  number  of  cysts  was  increasing  when  the  last  two 
examinations  were  made.  It  will  be  seen  that  there  are  not  two  com- 
plete periods  when  cysts  were  found  in  the  faeces,  and  because  of  this 
fact  these  data  of  rat  15  could  not  be  used  to  determine  the  cycle  of 
encystment  in  Giardia  of  the  rat. 

The  series  of  graphs  just  presented  may  be  divided  into  two  groups. 
In  the  first  group  may  be  placed  those  curves  which  show  positive 


Boech:  Studies  on  Giardia  Microti 


101 


examinations  comprising  less  than  one-half  the  total  number  of  exam- 
inations. The  graphs  of  figures  3,  4,  6,  12,  and  14  fall  into  this  class. 
In  the  second  group  may  be  placed  all  the  other  groups ;  in  these  more 
than  one-half  the  total  number  of  examinations  were  positive. 

What  caused  this  great  difference  in  the  degree  of  the  infection 
in  the  rats  is  not  known  at  the  present  time  for  there  is  no  evidence 
at  hand  with  which  to  attack  this  problem ;  but  it  is  very  significant 
that,  even  when  the  positive  examinations  comprised  less  than  half 
the  number  of  all  the  examinations  which  were  made,  there  are  still 
distinct  periods  into  which  the  positive  examinations  fall. 


3      4      5      6     7 


10    11    12    13   14    15 


Fig.  15 


Frequency  curve  of  modal  intervals. 

Curve  (solid)  plotted  by  single-day  units  (on  abscissa)  of  the  day  intervals 
between  the  modes  of  all  the  figures  1,  2,  4,  etc. 

Curve  (broken)  plotted  by  two-day  units,  of  the  day  intervals  between  modes 
of  all  the  curves.     It  is  a  more  typical  frequency  curve. 

The  interval  (average)  between  modes  is  shown  to  be  seven  days.  Seven 
days  is  the  interval  then,  between  the  maxima  number  of  cysts  in  the  faeces. 

This  graph  represents  the  combined  plot  of  the  intervals  between 
the  modes  of  all  the  curves  described  in  the  preceding  pages.  The 
units  on  the  abscissa  stand  for  the  length,  in  days,  of  each  interval; 
the  units  on  the  ordinate  stand  for  the  frequency  with  which  any 
given  interval  occurred. 

The  solid  line  is  a  frequency  curve  whose  descent  is  characteris- 
tically lytic  in  nature ;  the  latter  feature  is  eradicated  if  only  the  out- 
side points  are  represented  in  the  curve.  The  broken  line  curve  was 
made  from  the  same  data  as  the  solid  line  curve  except  that  the  units 
on  the  abscissa  are  two  days  in  length  instead  of  a  single  day.     This 


102  University  of  California  Puhlications  in  Zoology       [yoh.  19 

doubling  of  the  units  on  the  abscissa  results  in  the  curve  being  one- 
half  as  long  on  the  abscissa,  while  the  height  of  the  mode  is  far  more 
increased;  furthermore,  it  serves  to  straighten  out  the  descent  of  the 
curve,  giving  a  more  typical  frequency  graph. 

This  graph  presents  striking  proof  of  the  existence  of  a  cycle  of 
encystment  in  Giardia  in  the  rat,  for  we  see  that  the  average  interval 
between  the  modes  of  the  cycles  of  encystment  is  about  seven  days. 
Most  of  the  intervals  were  about  six  days  in  length.  In  other  words, 
the  maximum  number  of  cysts  were  found  in  the  faeces  about  every 
seven  days.  This  curve  indicates  that  encystment  falls  into  regular 
periods,  the  climax  of  each  recurring  period  being  reached  about  every 
seventh  day. 

If  a  series  of  curves  be  so  placed  one  above  the  other  that  the 
first  mode  of  each  curve  lies  on  the  same  ordinate  line  and  the  rest 
of  the  curve  be  allowed  to  fall  as  it  will,  if  there  is  a  similarity  or  a 
close  identity  of  the  interval  between  the  other  modes  of  each  curve, 
the  modes  of  all  the  curves  should,  in  the  majority  of  the  cases,  fall 
along  as  many  common  ordinate  lines  as  there  are  modes.  Since  most 
of  the  curves  have  three  or  four  modes  we  should  be  able  by  such  a 
handling  of  the  graphs  as  that  given  above  to  detect  four  common 
ordinate  lines  upon  which  the  majority  of  all  the  modes  of  the  curves 
will  be  located.  The  distance  between  these  lines  would  be  the  true 
interval  between  the  crest  of  each  mode,  or  the  interval  between  the 
days  when  the  maximum  numbers  of  cysts  were  found  in  the  faeces. 

Such  a  treatment  of  the  curves  is  the  most  conclusive  proof  of  the 
presence  of  similar  intervals  common  for  all  the  curves.  Obviously 
a  high  degree  of  exactness  is  impossible  with  the  curves  made  from  a 
study  of  the  cycle  of  encystment  in  rats,  since  the  incidence  of  in- 
fection varies  in  the  case  of  each  rat  and  there  is  also  a  margin  of 
error  in  the  detection  of  the  infection.  However,  there  is,  as  we  have 
seen,  evidence  for  an  interval  between  the  ejection  of  the  maximum 
numbers  of  cysts,  and  this  interval  can  be  seen  by  superimposing  the 
curves  one  above  the  other  with  their  first  modes  coinciding,  in  order 
to  determine  whether  or  not  the  other  modes  in  the  curves  will  in 
the  majority  of  cases  also  coincide  on  other  common  ordinates. 

In  figure  16  the  curves  have  been  placed  one  above  the  other 
so  that  their  first  modes  lie  on  the  ordinate  line  at  point  6  on  the 
abscissa.  In  this  figure  of  superimposed  curves  it  will  be  seen  that 
the  second  mode  for  the  majority  of  the  curves  is  on  an  ordinate  line, 
at  a  place  between  points  11  and  12  or  13  on  the  abscissa;  the  third 


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1919]  Boeck:  Studies  on  Giardia  Microti  103 

mode  of  those  curves  having  three  or  more  modes  lies  on  the  ordinate 
line  in  the  region  of  points  19  and  20  on  the  abscissa,  while  the  fourth 
mode  of  the  curves  which  have  four  modes  is  seen  to  lie  on  the  ordinate 
line  at  points  25  or  26  on  the  abscissa. 

In  all  cases  these  ordinate  lines  determined  above  were  selected 
because  the  majority  of  the  modes  of  all  the  curves  fell  on  these  four 
lines.  Thus  ten  curves  had  at  least  two  modes,  and  of  these  ten  six 
fell  on  the  ordinate  line  in  the  region  of  points  11,  12,  or  13  on  the 
abscissa.  Five  curves  showed  a  distinct  mode  in  the  region  of  points 
19  and  20  on  the  abscissa,  and  eight  curves  showed  definite  modes  on 
the  ordinate  line  in  the  region  of  points  25  and  26  on  the  abscissa. 
From  this  evidence  it  is  justifiable  to  conclude  that  there  is  a  common 
interval  between  the  modes  of  all  the  curves  when  they  are  placed 
one  above  the  other  so  that  their  first  modes  lie  upon  a  common 
ordinate  line.  This  interval  is  approximately  equal  between  all  the 
ordinate  lines.  The  intervals  between  the  ordinate  lines  are  6,  7,  and 
7  days;  their  average  interval  is  6%  days. 

The  average  interval  of  6%  days  as  obtained  from  the  super- 
imposition  of  the  curves,  is  almost  identical  with  the  average  interval 
of  7  days  obtained  by  plotting  the  frequency  of  all  the  modes  (see 
^g.  15).  Thus  by  two  different  methods  approximately  the  same 
interval  has  been  obtained,  setting  the  interval  between  the  maximum 
numbers  of  cysts  ejected  in  the  faeces  at  about  every  seven  days. 
In  other  words,  the  cycle  of  encystment  of  Giardia  occurred  about 
every  seven  days  in  the  rats  under  observation  during  a  period  of 
nearly  one  month. 

In  the  superimposition  of  the  curves  and  in  the  consequent  study 
of  the  data  to  determine  the  common  ordinate  lines  no  consideration 
was  taken  of  the  terminal  point  of  all  the  curves,  because  the  curve  was 
in  the  process  of  forming  another  mode  in  most  cases  when  our  obser- 
vations were  suspended.  Therefore,  because  of  its  incompleteness  this 
portion  of  the  curve  was  useless  in  the  determination  of  the  cycle  of 
encystment. 

In  order  to  substantiate  the  evidence  for  these  common  ordinate 
lines  which  mark  the  days  when  the  maximum  number  of  cysts 
occurred  in  the  faeces  of  most  of  the  rats  examined  I  plotted  the 
occurrence  of  the  average  number  of  cysts  for  each  day  when  the 
curves  were  thus  superimposed.  This  is  the  average  nujuber  of  cysts 
that  occurred  on  each  ordinate  line  in  figure  17.  For  example,  on 
ordinate  line  4  in  figure  16  two  cysts  were  found  twice,  three  cysts 


104  University  of  California  Fuhlications  in  Zoology       [^^ol.  19 

twice,  and  four  cysts  once.  The  average  number  of  cysts  on  this 
ordinate  was  three  cysts,  and  this  was  the  number  plotted  in  figure 
18  on  ordinate  line  5  as  representing  the  average  number  of  cysts  for 
that  day.  In  like  manner  the  average  number  of  cysts  was  determined 
for  the  other  days  and  plotted  in  figure  17. 

The  modes  1,  2,  and  4  are  at  once  very  evident  and  it  will  be  found 
that  they  lie  on  the  same  ordinate  lines  that  were  determined  in 
figure  16. ,  The  third  mode  is  not  so  conspicuous,  which  I  interpreted 
to  mean  that  the  incidence  of  encystment  was  small  and  disturbed, 
possibly  by  some  environmental  factor  for  all  the  rats  whose  modes 
go  to  make  up  this  common  mode.  But  it  will  be  seen,  as  w^as  pointed 
out  in  connection  with  figure  16,  that  6  rats  showed  a  distinct  mode 
at  this  area  in  their  curve.  Of  the  other  six  rats  one  was  showing  a 
small  number  of  cysts  at  this  time  (fig.  5),  three  were  negative,  and 
no  cysts  were  found  in  the  following  examinations  (figs.  4,  6,  12),  so 
these  curves  could  not  be  considered ;  the  other  two  rats  were  negative 
during  these  days.  Therefore,  out  of  eight  possible  cases  six  of  them 
showed  a  distinct  mode  at  point  19  (fig.  17),  which  made  it  justifiable 
to  pick  out  this  line  as  representing  a  common  mode  for  most  of  the 
curves.    It  was  the  day  when  a  maximum  number  of  cysts  was  found. 

The  mode  at  26  (fig.  17)  was  chosen  rather  than  the  mode  at  28 
because  more  individual  curves  showed  a  distinct  mode  at  this  day, 
and  so  represented  more  truly  the  day  when  the  maximal  number  of 
cysts  was  found  for  the  majority  of  all  the  rats. 

The  average  interval  between  the  modes  1,  2,  3,  and  4  (fig.  17) 
is  found  to  be  6%  days,  which  corresponds  to  the  interval  between 
the  ordinate  lines  determined  in  figure  16. 


Discussion 

From  the  data  just  presented  the  conclusion  is  derived  that  there 
is  a  cycle  of  encystment  in  Giardia  in  the  rat,  that  this  cycle  is  regu- 
larly periodic,  and  that  the  interval  between  successive  maximum 
numbers  of  cysts  is  about  seven  days. 

It  is  fully  realized  that  the  possibility  of  error  is  fairly  large  in 
the  study  that  has  been  made  in  determining  this  cycle.  In  the  first 
place  because  of  the  time  required  for  examination  only  one  examina- 
tion was  made  to  determine  whether  or  not  a  rat  was  negative  for  any 
given  day.  To  eliminate  this  error  at  least  three  ordinary  examina- 
tions should  be  made  to  determine  whether  a  rat  is  negative.    Had  it 


1919] 


Boeck:  Studies  on  Giardia  Microti 


105 


been  possible  to  continue  the  examinations  by  means  of  the  ether- 
concentration  method,  adopted  later,  then  three  ordinary  examinations 
would  not  have  been  necessary,  one  examination  by  this  method  would 
have  sufficed. 

Again,  in  all  the  studies  of  this  nature  the  more  cases  one  works 
with  the  safer  are  his  conclusions;  therefore,  if  a  greater  number  of 
rats  could  have  been  handled  the  conclusions  would  have  a  greater 
degree  of  certainty.  But  even  with  this  relatively  small  number  of 
cases,  when  the  results  are  to  a  great  extent  uniform,  the  conclusions 
are  at  least  highly  significant  and  to  a  considerable  degree  may 
be  relied  on  as  presenting  the  truth  of  the  situation.     The  data  from 


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Fig.  18 


Frequency  curve  of  mode  intervals;  data  taken  from  charts  submitted  by 
Porter   (1916). 

Solid  line,  interval  plotted  by  single-day  units,  modes  at  8,  15,  24, 

Broken  line,  interval  plotted  by  two-day  units,  modes  at  6,  9,  14. 

The  broken  line  curve,  a  typical  bi  modal  polygon. 

The  interval  between  the  maximal  number  of  cysts  is  about  7-8  days.  Four- 
teen days  represents  two  intervals,  and  the  small  mode  at  24  represents  three 
intervals. 

These  results  from  studies  of  seven  cases  of  Giardia  intestinalis  are  very 
similar  to  those  given  by  me  for  G.  microti. 

the  fourteen  rats  presented  by  several  methods  yield  in  every  case  the 
same  fairly  uniform  and  equivalent  cycle  of  encystment. 

Another  source  of  evidence  is  available  for  comparison  to  demon- 
strate the  value  of  these  results  in  this  small  number  of  cases.  In 
determining  the  cycle  of  encystment  for  Giardia  intestinalis  from 
man  and  mice  Porter  (1916)  noticed  that  there  was  a  period  of  about 
a  fortnight  between  the  maximum  number  of  cysts  recurring  in  the 
faeces.  She  did  not,  however,  call  attention  to  the  fact  that  there 
was  also  a  period  of  about  seven  or  eight  days  when  another  maximum 
number  of  cysts  was  to  be  found  in  the  faeces.  This  she  would  have 
found  apparently  if  the  day-interval  between  the  modes  in  all  her 
figures  had  been  plotted. 


106 


University  of  California  Publications  in  Zoology        [Vol.  19 


It  is  significant  that  a  plot  made  of  analogous  data  in  my  own 
study  reveals  only  one  mode  (fig.  15)  and  sets  the  period  at  seven  days 
as  the  interval  between  maximum  numbers  of  cysts  in  the  faeces. 
The  curve  is  a  typical  frequency  curve  with  a  single  mode. 

In  plotting  the  interval  between  the  modes  in  the  curves  sub- 
mitted by  Porter  (1916)  there  are  two  distinct  modes  present  (fig.  18). 
It  is  a  bimodal  frequency  curve.  The  solid  line  in  figure  18  repre- 
sents the  interval  plotted  by  single-day  units  on  the  abscissa,  just  as 
was  done  in  figure  15  of  my  own  study.  Two  distinct  modes  are  seen 
at  points  8  and  15  and  a  small  mode  at  point  24.  There  was  only  one 
instance  of  an  interval  of  24  days  (see  chart  1,  Porter,  1916).  When 
these  same  data  were  plotted  by  two-day  units  on  the  abscissa,  the 


1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31 

Fig.  19 

A  plot  of  the  average  number  of  cysts  for  each  day  for  rats  1,  2,  4,  5,  6,  8,  9, 
10,  11,  12,  13,  14,  leaving  out  of  consideratoin  the  number  of  cysts  which  occurred 
on  the  last  examination  in  each  rat. 

Modes  1,  2,  and  4  are  very  definite;  mode  3,  although  not  so  pronounced  as 
the  other  modes,  was  selected  since  in  six  rats  this  point  represented  the  days  of  a 
maximal  number  of  cysts  in  the  faeces.  Mode  4  at  point  27  on  the  abscissa  was 
selected  instead  of  the  mode  at  point  29  because  it  was  more  representative  of 
all  the  curves;  five  curves  had  a  mode  at  or  near  point  27,  while  only  two  curves 
had  a  mode  at  point  29. 

The  average  day-intervals  between  modes  1,  2,  3,  4,  is  6%  days  are  almost 
identical  with  the  intervals  found  in  figure  15. 

broken  curve  results,  and  again  we  find  that  there  are  two  modes 
present.  This  curve  thus  plotted  represents  two,  if  not  three  (point 
24),  distinct  periods  at  which  time  maximum  numbers  of  cysts  were 
found  in  the  faeces  for  Giardia  intestinalis. 

From  this  study  of  Porter's  charts  and  the  plottings  of  the  modes 
of  her  graphs  it  is  evident  that  the  cycle  of  encystment  in  G.  intes- 
tinalis has  an  interval  of  about  seven  to  eight  days  between  the  maxi- 
mum number  of  cysts  in  the  faeces.  The  mode  at  point  24  (fig.  18)  is 
the  locus  of  a  third  occurrence  in  one  instance  only  of  a  maximum  num- 
ber of  cysts,  which  suggests  that  there  was  a  tendency  at  least  for  the 
cycle  to  continue  at  the  second  interval  of  fourteen  days  on  the  interval 


1919]  '  Boeck:  Studies  on  Giardia  Microti  107 

of  the  first  two  maxima.  The  results  are  very  striking  and  uniform 
in  this  series  of  only  seven  cases,  and  taken  together  with  the  data 
derived  from  my  study  of  the  cycle  of  encystment  in  Giardia  of  the  rat 
give  ground  for  concluding  that  there  is  a  regular  periodicity  in  the 
appearance  of  the  cysts  of  Giardia  in  the  faeces  of  about  six  to  seven 
days. 

It  is  quite  possible  that  the  species  of  Giardia  found  in  some  of 
the  rodents,  especially  in  G.  microti,  is  one  and  the  same  species  as 
that  found  in  man,  namely,  G.  intestinalis,  because  of  the  similarity 
in  structure.  Another  reason  for  believing  that  these  species  may  be 
identical  is  revealed  by  the  study  of  periodicity  made  by  myself  on 
G.  microti  and  by  Porter  (1916)  on  G.  intestinalis,  and  also  by  the 
manipulation  of  her  data  in  the  plotting  of  the  curve  representing 
the  frequency  of  the  interval  between  modes,  or  the  days  when  the 
maximum  number  of  cysts  was  detected.  This  study  shows  that  both 
of  these  species  have  almost  identical  cycles  of  encystment.  This 
feature  may  be  common  for  all  the  species  of  Giardia,  therefore  a 
characteristic  of  the  genus  and  not  a  peculiarity  of  a  single  species 
only ;  but  in  either  case  it  is  significant  as  evidence  toward  the  solution 
of  the  true  etiology  of  giardiasis  (lambliasis)  of  man. 


Summary 

1.  There  is  a  cycle  of  encystment  in  Giardia  in  the  rat ;  the  recur- 
ring interval  between  the  maximum  numbers  of  cysts  in  the  faeces  is 
about  seven  days. 

2.  The  cycle  of  encystment  in  G.  intestinalis  is  about  seven  or 
eight  days,  as  seen  in  the  charts  submitted  by  Porter  (1916),  and  not 
a  fortnight,  as  she  has  concluded.  This  in  reality,  if  not  fully  plotted, 
represents  two  cycles  of  encystment. 

3.  Because  the  cycles  of  encystment  of  Giardia  in  the  rat  and  of 
G.  intestinalis  are  almost  identical  there  is  another  reason  to  infer 
that  these  two  species  may  be  one  and  the  same.  This  fact  may  lend 
some  aid  in  the  solution  of  the  true  etiology  and  prevention  of 
giardiasis  (lambliasis)  of  man. 


108  TJniversity  of  California  Piiblications  in  Zoology        [Yoh.  19 

DEVELOPMENT  OF  GIARDIA  MICROTI  WITHIN  THE  CYSTS 

Introduction 

The  author  (1917)  has  pointed  out  the  presence  of  three  distinct 
types  of  cysts  for  Giardia  microti.  These  types  were  placed  in  three 
categories  because  their  morphology  warranted  such  a  classification. 
The  first  of  these  types  is  the  ''single  individual"  cyst  (pi.  1,  fig.  1). 
This  cyst  harbors  only  one  individual  which  has  the  usual  two  nuclei. 
The  second  type  is  the  "binary"  cyst  (pi.  1,  figs.  5-8),  containing  four 
nuclei.  The  cytoplasmic  body  may  not  have  undergone  plasmotomy, 
(pi.  1,  figs.  4,  5)  or  plasmotomy  may  be  completed  with  two  daughter 
flagellates  formed  (pi.  1,  figs,  7,  8).  The  third  type  is  the  ''multinu- 
cleate" cysts,  so  called  because  it  always  contains  more  than  four 
nuclei  and  never  more  than  sixteen  nuclei  (pi.  1,  figs.  12-16). 

A  study  of  these  cysts  and  their  location  in  the  intestine  of  the 
mouse  or  rat  at  once  suggest  definite  progressive  development  of  the 
flagellate  within  the  cyst.  To  give  an  account  of  this  development 
with  a  view  to  supptying  more  evidence  toward  completing  all  the 
stages  in  the  life  history  of  this  parasite,  the  following  data  are 
submitted. 

Materials  and  Data 

The  first  part  of  these  data  were  gathered  from  a  study  of  the 
material  from  ^yq  different  meadow  mice,  Microtus  calif ornicus  cali- 
fornicus  (Peale).  Other  preparations  were  made  but  only  five  series 
revealed  the  presence  of  cysts.  The  following  table  shows  the  types 
of  cysts  found  in  the  different  regions  of  the  digestive  tract  of  the 
meadow  mice. 

Cysts  were  not  always  found  in  all  the  regions  of  the  digestive  tract 
(series  28,  A4)  and  this  was  explained  by  virtue  of  a  cycle  of  encyst- 
ment.  In  series  28  the  process  of  encystment  had  been  going  on  for 
some  time  and  the  cysts  had  already  progressed  as  far  as  the  colon  and 
rectum,  while  in  series  A4,  the  process  of  encystment  had  just  begun 
and  so  the  cysts  were  found  only  in  the  small  intestine.  In  series  29 
and  33  preparations  made  from  all  the  regions  of  the  intestine  showed 
the  presence  of  cysts.  Encystment  was  in  the  midst  of  its  cycle. 
Series  29  and  33  present  two  parallel  lines  of  data,  identical  in  all 
respects  except  that  in  series  29  only  single  and  binary  cysts  were 


1919]  Boeck:  Studies  on  Giardia  Microti  109 


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110  University  of  California  PuUications  in  Zoology       L^ol.  19 

found,  while  in  series  33  not  only  were  the  single  and  binary  cysts 
present  but  also  multinucleate  cysts.  This  latter  type  of  cyst  was 
found  chiefly  in  the  small  intestine. 

The  probable  reason  why  the  cysts  were  found  in  so  few  of  the 
infected  mice  at  autopsy  was  the  fact  that  a  negative  period  in  the 
cycle  of  encystment  was  then  in  progress. 

In*  Giardia  microti  there  are  two  types  of  reproduction,  binary  and 
multiple  fission.  In  taking  up  the  evidence  for  the  development  of 
this  flagellate  within  the  cysts  a  review  will  be  made  first  of  the  data 
that  are  related  to  the  reproductive  method  by  binary  fission  and 
then  the  data  concerned  with  the  method  of  multiple  fission. 

Binary  fission  may  occur  in  the  free  state  of  the  flagellate,  as  has 
been  shown  by  the  work  of  Kofoid  and  Christiansen  (1915)  and  by 
the  author  (1917),  and  also  within  the  cyst;  the  latter  fact  has  been 
known  for  a  long  time.  Previous  to  the  work  mentioned  above,  de- 
scribing binary  fission  taking  place  in  the  free  state,  it  had  been  held 
that  this  method  of  fission  took  place  only  within  the  cyst.  Schaudinn 
(1903)  had  noticed  two  individuals  within  a  single  cyst  wall  and  had 
called  such  a  cyst  a  "copulation  cyst"  because  he  thought  the  two 
flagellates  were  in  syngamous  union.  It  had  been  previously  shown, 
however  (Boeck,  1917),  that  these  cysts  were  only  binary  cysts  and 
that  there  is  no  evidence  of  sex  in  Giardia  as  Schaudinn  had  inferred. 

Binary  Fission  Within  the  Cyst 
When  encystment  takes  place  it  involves  the  formation  of  a  wall 
around  a  single  flagellate.  This  process  begins  in  the  ileum  and 
caecum  of  the  meadow  mouse.  The  greatest  number  of  single  cysts 
are  consequently  found  in  the  ileum  or  caecum  of  the  digestive  tract. 
From  table  2  it  will  be  seen  that  these  single  cysts  may  also  be  found 
in  the  colon  and  rectum,  but  their  number  in  these  regions  is  very 
small  compared  with  the  number  found  in  the  small  intestine.  The 
ratio  in  series  29  in  one  preparation  was  fifty-five  binary  cysts  to  five 
single  individual  cysts  in  the  colon  and  rectum. 

The  fact  that  there  were  great  numbers  of  single  cysts  in  the  ileum 
and  only  a  few  in  the  large  intestine,  great  numbers  of  binary  cysts 
being  present  in  their  stead,  coupled  with  the  presence  of  nuclear 
changes  within  the  cysts,  is  conclusive  evidence  of  progressive  multi- 
plication within  the  cysts. 

The  single  cyst  (pi.  1,  fig.  1)  is  strongly  indicative  of  recent  encyst- 
ment because  one  can  easily  detect  within  it  all  the  organelles  of  the 


1919]  BoecJc:  Studies  on  Giardia  Microti  111 

flagellate  characteristic  of  its  free  state,  the  only  exception  being 
the  absence  of  the  extracytoplasmic  parts  of  the  anterolateral,  postero- 
lateral, and  caudal  flagella.  The  intracytoplasmic  portions  of  these 
last  named  structures  are  still  present  within  the  cyst. 

In  the  metamorphosis  of  a  single  individual  cyst  into  a  binary 
cyst  the  nuclei  undergo  division.  Distinct  anaphase  spindles  were 
found  in  many  cases  (pi.  1,  fig.  3),  but  no  late  prophase,  metaphase,  or 
telophase  stage  was  found.  The  mitotic  activity  during  these  stages 
is  probably  very  rapid  while  the  anaphase  is  of  longer  duration. 
The  telophase  was  detected  in  cysts  of  Giardia  found  in  the  rats 
(pi.  1,  fig.  12).  In  no  instance  was  the  chromatin  of  either  nucleus 
seen  to  have  resolved  itself  into  chromosomes.  Centrosomes  were  dis- 
tinguishable in  some  of  the  cysts  (pi.  1,  figs.  2,  4),  but  in  no  case  were 
they  found  to  be  divided  with  one  daughter  centrosome  located  at 
each  pole  of  each  nucleus. 

In  the  cyst  shown  in  plate  1,  figure  1,  the  nuclei  of  the  organism 
appear  to  be  in  the  resting  stage,  for  the  karyosome  is  a  single  ovoid 
mass  of  chromatin.  This  stage  in  the  development  within  the  cyst 
is  very  typical  of  the  nuclear  constitution  of  all  the  single-individual 
cysts  found  in  the  ileum  and  the  caecum. 

In  the  caecum  single-individual  cysts  were  also  found,  which, 
however,  present  the  first  stage  of  definite  progressive  development 
beyond  that  described  in  the  preceding  paragraph.  These  cysts 
(pi.  1,  figs.  2-4)  show  the  chromatin  of  the  nuclei  dividing  or  already 
divided  into  two  separate  masses  upon  a  spindle,  with  evidence  of  their 
migration  to  the  poles  of  each  nucleus.  This  stage  corresponds  to  the 
anaphase  in  mitosis.  Passing  along  the  digestive  tract  from  the 
caecum  to  the  colon  we  find  a  few  of  the  single-individual  cysts  are 
still  to  be  found,  even  as  far  down  as  the  rectum,  but  their  number 
is  very  small. 

In  the  colon  the  next  stages  of  development  within  the  cysts  were 
found.  The  nuclei  have  divided  to  form  four  daughter  nuclei  (pi.  1, 
fig.  5),  which  are  very  close  to  each  other  and  in  the  resting  stage. 
The  cytoplasmic  body  within  the  cyst  is  still  in  the  condition  of  a 
single  encysted  individual,  no  plasmotomy  having  as  yet  taken  place. 
The  axostyle  has  partially  divided,  but  only  two  distinct  parabasal 
bodies  are  to  be  seen. 

The  next  stage  in  the  progress  of  binary  fission  within  the  cyst 
is  also  to  be  found  in  the  cysts  in  the  colon.  In  this  stage  (pi.  1,  fig.  6) 
two  of  the  nuclei  have  migrated  to  a  position  which  was  earlier  the 


112  University  of  California  Puhlications  in  Zoology       [Vol.  19 

posterior  pole  of  the  body  of  the  parent,  but  no  plasmotomy  has  as  yet 
taken  place.  During  this  stage  another  set  of  intracytoplasmic 
organelles,  namely  axostyle,  anterior  peristomal  fibrils,  and  postero- 
lateral flagella  have  been  formed.  The  method  of  forming  the  axo- 
style is  one  of  splitting  of  the  parent  structure  (pi.  1,  fig.  3),  but 
there  is  no  evidence  at  hand  to  determine  the  exact  method  of  forma- 
tion of  the  other  organelles. 

In  the  colon  and  in  the  rectum  occur  the  binary  cysts  (pi.  1, 
figs.  7,  8)  which  represent  the  completion  of  binary  fission.  The 
method  of  plasmotomy  appears  to  be  a  longitudinal  division  of  the 
parent  on  a  plane  parallel  to  the  major  axis  of  the  body  and  at  first 
horizontal,  at  least  in  the  matter  of  nuclear  separation,  for  even  before 
plasmotomy  has  begun  (pi.  1,  fig.  6)  two  of  the  nuclei  and  the  axostyle 
are  seen  to  be  upon  a  different  optical  plane  from  that  of  their  sister 
structures.  The  axostyle,  however,  appears  to  split  in  the  sagittal 
plane.  When  plasmotomy  has  been  completed  a  side  view  of  the  two 
flagellates  within  the  cyst  shows  one  individual  above  the  other.  The 
line  of  separation  of  the  two  flagellates  may  have  no  reference  to  any 
plane  of  plasmotomy.  Kofoid  and  Swezy  (1916)  have  shown  that 
movement  in  trichomonads  is  very  active  during  plasmotomy  in  the 
free  state.  It  is  quite  possible  that  this  also  occurs  within  the  cyst 
in  Giardia.  An  oblique  view  (pi.  1,  fig.  8)  shows  the  two  fiagellates 
occupying  such  a  position  that  their  anterior  ends  are  at  opposite  ends 
of  the  cyst ;  this  is  the  position  which  they  might  assume  if  plasmotomy 
occurred  by  a  longitudinal  cleavage  of  the  body  in  a  plane  horizontal 
to  the  major  axis  of  the  body.  However,  in  typical  binary  cysts  the 
two  zooids  lie  in  an  end  to  end,  back  to  back  position. 

It  is  noteworthy  that  in  all  the  cysts  of  the  binary  type  the  indi- 
viduals resulting  from  binary  fission  of  the  parent  flagellate  lack 
their  respective  parabasal  bodies.  This  absence  has  been  shown  by 
the  author  (1917)  to  be  correlated  with  the  depletion  of  the  reserve 
material  in  the  form  of  these  bodies  during  encystment,  and  with 
excessive  mitotic  and  motor  activity  of  the  flagellate  while  in  the  free 
state.  These  bodies  at  first  hypertrophy  during  encystment,  ofteji 
they  are  scattered  like  a  cloud  of  chromotoidal  substance  in  the  cyto- 
plasm (Kofoid  and  Christiansen,  1915),  but  are  always  absent  in  the 
free  somatella  stage  or  when  plasmotomy  during  binary  fission  within 
the  cyst  has  been  completed. 

Infection  is  known  to  occur  by  the  ingestion  of  the  cysts ;  no  inter- 
mediate host  is  necessary.    The  cyst  wall  in  all  probability  is  digested 


1919]  Boech:  Studies  on  Giardia  Microti  113 

off  when  the  cysts  reach  the  small  intestine.  Those  cysts  which  con- 
tain two  individuals,  products  of  binary  fission,  would  discharge  two 
flagellates  into  the  intestine  when  the  cyst  wall  was  digested  away. 
It  is  also  possible  that  cysts  might  be  ingested  which  had  not  completed 
all  the  stages  of  binary  fission,  because  such  cysts  were  found  in  the 
rectum,  and  if  this  happened  and  the  cyst  wall  was  digested  away 
then  a  somatella  would  be  liberated  which  could  continue  its  develop- 
ment in  a  free  stage. 

A  word  may  be  said  here  regarding  these  somatella  stages  de- 
scribed many  times  by  Kofoid  and  Christiansen  (1915)  for  G.  muris 
and  by  the  same  investigators  and  myself  for  G.  microti.  Most  of 
these  somatella  stages  represent  plasmodial  bodies  resultant  from 
previous  mitotic  activity  of  individuals  which  had  not  encysted. 
Obviously  the  large  number  of  individuals  present  in  the  intestine 
can  be  explainable  on  the  ground  of  binary  and  multiple  fission  in 
the  free  stage  of  the  flagellate,  but  another  interpretation  may  be 
proposed  for  the  somatella  stages. 

These  stages  may  be  interpreted  to  be  somatellas  liberated  from 
cysts  while  the  flagellate  body  within  w^as  in  the  course  of  binary 
fission.  Especially  does  this  seem  plausible  when  we  compare  the 
free  somatella  described  by  Kofoid  and  Christiansen  (pi.  8,  fig.  55; 
1915)  and  the  encysted  somatella  described  in  this  paper  (pi.  1,  fig.  6). 
Furthermore,  these  authors  figure  cysts  with  part  of  the  cyst  wail 
apparently  digested  away  and  the  somatella  in  a  process  of  binary 
fission  (pi.  7,  fig.  38;  Kofoid  and  Christiansen,  1915).  The  interpre- 
tation that  these  somatellas  may  have  originated  from  encysted  indi- 
viduals which  have  escaped  from  the  cyst  seems  justifiable  in  view 
of  the  facts  just  presented.  But  most  of  these  somatella  stages  no 
doubt  originate  from  an  individual  undergoing  binary  fission  in  the 
free  state. 

Multiple  Fission  Within  the  Cysts 
During  the  process  of  multiple  fission  within  the  cysts  the  two 
nuclei  of  the  encysted  flagellate  go  through  three  successive  divisions 
to  form  a  total  of  sixteen  daughter  nuclei.  The  method  of  division 
is  that  of  mitosis,  for  anaphase  spindles  were  found  in  many  cases 
(pi.  1,  fig.  10),  but  the  other  phases  of  mitosis  were  much  obscured 
and  were  therefore  detected  with  gerat  difficulty.  The  chromatin  of 
each  nucleus  was  never  seen  to  be  divided  into  chromosomes.  A  soma- 
tella containing  sixteen  nuclei  is  found  in  every  multinucleate  cyst 
which  has  completed  all  its  nuclear  divisions  (pi.  1,  figs.  10,  11). 


114  University  of  California  Puhlications  in  Zoology       [Vol.  19 

From  table  2  we  see  that  the  greater  number  of  these  cysts  were 
found  in  the  small  intestine,  and  in  one  series  they  were  found  in 
the  rectum.  This  was  interpreted  to  mean  that  encystment  com- 
menced in  the  small  intestine  (duodenum)  and  the  cysts  were  in  the 
rectum  because  they  had  been  carried  there  by  the  peristaltic  move- 
ment of  the  bowels. 

Encystment,  it  appears,  begins  with  the  formation  of  a  wall  around 
a  single  individual,  and  the  immediately  subsequent  stages  are  sim- 
ilar to  those  in  binary  fission  preceding  plasmotomy  of  the  two-zooid 
somatella.  Many  of  the  stages  following  the  first  division  of  the  nuclei 
were  not  found.  Some  of  the  cysts  showed  twelve  nuclei  (pi.  1,  fig.  9) 
due  to  the  fact  that  some  of  the  nuclei  have  not  divided  the  third  time. 
In  some  cysts  (pi.  1,  fig.  10)  two  of  the  nuclei  were  seen  to  be  dividing 
for  their  last  time  (third  division) .  No  plasmotomy  was  seen  to  occur 
within  the  cyst,  but  from  the  evidence  given  by  Kofoid  and  Christian- 
sen (1915)  it  is  more  probable  that  plasmotomy  takes  place  outside  the 
cysts  after  the  digestion  of  the  wall  of  the  cysts  in  the  small  intestine. 

This  method  of  reproduction  within  the  cysts  does  not  seem  to  be  the 
common  method  employed  by  this  flagellate,  for  the  cysts  were  detected 
in  only  two  cases  in  my  preparations  of  G.  microti  from  the  meadow 
mouse  and  were  never  detected  in  some  two  hundred  examinations 
of  cysts  of  a  species  of  Giardia  found  in  the  rat.  It  is  a  method 
which  results  in  a  greater  proliferation  of  the  individuals  when  com- 
pared with  binary  fission.  In  the  rat  the  binary  cysts  were  found  in 
the  faeces  at  regular  intervals,  showing  that  encystment  was  cyclic 
and  not  continuous  or  sporadic  in  nature.  But  since  the  multiple 
cysts  were  not  found  with  such  regularity  it  appears  that  this  method 
of  reproduction  does  not  follow  at  regular  intervals  unless  its  interval 
of  recurrence  is  much  longer  than  that  found  for  binary  fission.  It 
may  be,  then,  that  disturbance  of  the  normal  environment  causes  an 
increased  fecundity  on  the  part  of  the  flagellates  which  results  in  the 
method  of  multiple  fission  for  its  expression.  The  possibility  of 
multiple  fission  following  the  formation  of  a  zygote  by  syngamy  is  not 
excluded. 

These  cysts  when  ingested  would  have  their  cyst  walls  digested 
away  as  in  the  case  of  the  binary  cysts.  Since  each  one  of  the  multi- 
nucleate cysts  contained  a  somatella  of  sixteen  nuclei  an  eight-zooid 
somatella  would  be  liberated  to  continue  its  plasmotomy  in  a  free 
state.  Cysts  showing  part  of  the  cyst  wall  absent  were  figured  by 
Kofoid  and  Christiansen   (1915,  pi.  7,  figs.  42,  53)   and  these  cysts 


1919]  Boeck:  Studies  on  Giardia  Microti  115 

contained  somatellas  in  various  stages  of  multiple  fission.  The  absence 
of  part  of  the  cyst  wall  is  interpreted  to  be  due  to  digestion  and  a 
continuance  of  the  process  would  result  in  the  liberation  of  the  soma- 
tella.  Somatella  comprising  as  many  as  four  zooids  were  found  by  the 
investigators  just  mentioned,  but  in  no  case  were  eight-zooid  soma- 
tellas found.  The  case  of  the  four-zooid  somatella  appears  to  have 
resulted  from  the  multiple  fission  of  a  flagellate  which  had  never 
encysted,  instead  of  representing  a  possible  half  of  the  eight-zooid 
individual  which  might  have  come  from  a  cyst. 

In  all  the  multinucleate  cysts  the  axostyle  had  split  to  form  two 
daughter  axostyles.  This  is  similar  to  the  activity  of  the  parent  axo- 
style during  binary  fission,  but  the  other  organelles  were  not  duplicated 
which  is  unlike  the  condition  in  binary  fission.  It  is  probable  that 
the  formation  of  the  other  organelles  is  deferred  until  the  process  of 
plasmotomy  begins,  that  being  the  time  that  they  appear  in  binary 
fission. 

It  is  evident  from  all  the  data  at  hand  that  the  processes  of  binary 
and  multiple  fission  are  very  similar  if  not  identical  in  their  early 
stages.  Both  methods  begin  with  the  encystment  of  a  single  individual, 
and  the  subsequent  stages  up  to  the  formation  of  a  two-zooid  somatella 
are  alike.  The  process  of  binary  fission  ceases  when  plasmotomy  of  the 
two-zooid  somatella  results  in  the  production  of  two  daughter  flagel- 
lates; but  with  the  formation  of  the  two-zooid  somatella  the  process 
of  multiple  fission  still  goes  on.  The  nuclei  have  gone  through  one 
division  to  form  four  nuclei,  the  four  nuclei  go  through  another 
division  and  eight  nuclei  are  formed,  and  these  eight  nuclei,  when 
they  divide,  form  sixteen  nuclei.  There  are  three  successive  divisions 
of  the  nuclei  in  multiple  fission  but  only  one  division  in  binary 
fission.  The  two  processes  differ  in  that  multiple  fission  does  not 
appear  to  occur  so  often  as  binary  fission  since  the  cycle  of  encyst- 
ment during  which  binary  fission  takes  place  is  shorter  than  the  cycle 
of  multiple  fission,  if  a  cycle  of  multiple  fission  exists.  It  is  probable 
also,  as  has  been  stated,  that  multiple  fission  is  initiated  by  some 
environmental  disturbances  and,  therefore,  may  not  be  cyclic  but 
sporadic  in  nature. 

The  data  for  binary  and  multiple  fission  within  the  cysts  of 
Giardia  microti  were  taken  from  preparations  made  from  the  meadow 
mouse.  A  further  collection  of  data  will  now  be  presented  to  cor- 
roborate the  evidence  for  binary  and  multiple  fission  within  the  cyst. 
These  further  data  were  taken  from  a  study  of  the  cysts  of  a  species 


116  University  of  California  Publications  in  Zoology       ["Vol.  19 

of  Giardia  found  in  culture  rats.  This  species  of  Giardia  resembles 
G.  microti  as  far  as  the  general  form  of  the  body  and  the  organelles 
are  concerned.  The  species  is  longer  than  G.  microti,  measuring  13 
to  17  microns  in  length  and  6  to  9  microns  in  width.  The  cysts, 
however,  are  approximately  the  same  size  as  those  of  G.  microti;  they 
measure  11  to  13  microns  in  length  and  5  to  7  microns  in  width 
(compare  pi.  1,  figs.  5,  13). 

The  locus  of  infection  in  the  rat  differs  from  that  of  the  meadow 
mouse.  In  the  meadow  mouse  the  flagellates  were  found  in  all  the 
regions  of  the  small  intestine,  but  in  the  rat  the  jejunum  is  the  area 
that  is  most  heavily  infected.  No  flagellates  occurred  in  the  duodenum 
and  only  a  few  in  the  ileum.  Again,  it  was  found  that  the  intestine 
of  the  meadow  mouse  was  not  discolored  by  an  infection  with  Giardia 
(Boeck,  1917),  but  in  the  rat  the  jejunum  is  orange  colored  and  filled 
with  gas  when  Giardia  are  present.  This  latter  condition  is  very 
similar  to  the  condition  found  in  culture  mice  and  Peromyscus  gamheli 
when  infected  with  G.  muris. 

A  study  was  made  of  the  cysts  found  in  the  different  regions  of 
the  small  and  large  intestines  in  order  to  see  if  the  method  of  develop- 
ment for  this  species  of  Giardia  during  encystment  was  similar  to 
that  of  G.  microti  during  its  encystment. 

The  following  table  gives  the  types  of  cysts  found  in  the  different 
regions  of  the  digestive  tract  of  the  mouse. 


TABLE  3 

Types  of  cysts 

OF  Giardia  in  intestine  of  kat 

Series 

Sex 

Date 

Small  intestine 

Colon 

Rectum 

A5 

c? 

Oct.  12, 1917 

single  binary 

binary 

binary 

A6 

c^ 

Oct.  21, 1917 

binary 

binary 

200 

Feb.  8,  1918 

binary 

binary 

binary 

504 

Feb.  8,  1918 

binary 

binary 

307 

Feb.  8,  1918 

single  binary 

binary 

binary 

313 

Feb.  8, 1918 

binary 

binary 

binary 

It  will  be  seen  at  once  that  the  only  types  of  cysts  encountered 
in  the  preparations  were  the  single-individual  and  the  binary  cysts; 
no  multinucleate  cysts  were  ever  found.  In  the  meadow  mouse  the 
binary  cysts  did  not  occur  until  the  cysts  had  reached  the  colon  on 
their  transit  through  the  intestine  to  the  outside  of  the  mouse,  but  in 
the  rat  the  binar}^  cysts  occurred  in  the  jejunum  along  with  the 
flagellates  in  the  free  state.  The  single-individual  cysts  were  few  in 
number  compared  with  the  binary  cysts,  and  so  it  would  seem  that  the 


1919]  Boeck:  Studies  on  Giardia  Microti  117 

division  of  the  nuclei  to  form  four  daughter  nuclei  follows  soon  after 
the  encystment  of  the  flagellate.  The  cysts  are  defaecated  as  binary 
cysts,  for  over  two  hundred  preparations  from  the  faeces  were  exam- 
ined and  showed  the  cysts  at  a  stage  of  development  in  binary  fission 
the  same  as  that  found  in  the  small  intestine. 

In  the  rat  the  binary  cysts  occurred  throughout  the  large  intestine 
and  were  found  in  the  faeces  in  a  stage  of  development  which  was  not 
in  advance  of  the  stage  that  was  found  in  the  jejunum  and  ileum 
(pi.  ,  figs.  14,  16).  A  few  of  the  single-individual  cysts  were  also 
found  in  the  large  intestine,  but  the  number  was  almost  insignificant 
compared  with  the  number  of  binary  cysts.  The  single-individual 
cysts  of  Giardia  found  in  the  rat  resembled  very  closely  those  of 
G.  microti  which  were  found  in  the  meadow  mouse  (cf.  pi.  1, 
figs.  1,12). 

Mitosis,  with  its  phases  somewhat  modified,  is  the  method  of  nuclear 
division.  At  no  time  in  the  cysts  examined  could  chromosomes  be 
detected  during  the  process  of  mitosis.  Anaphase  spindles  (pi.  1, 
fig.  12)  were  very  common.  When  the  new  nuclei  are  formed  two 
remain  in  the  anterior  region  of  the  cyst;  but  the  other  pair 
appear  to  migrate  to  a  more  posterior  position,  and  they  lie  upon  a 
different  optical  plane  from  that  occupied  by  the  other  pair  (pi.  1, 
figs.  13-16).  This  difference  in  optical  planes  of  the  two  pairs  of 
nuclei  is  due  to  the  direction  of  the  major  axis  of  the  spindle  during 
the  division  of  the  parent  nuclei.  The  major  axis  of  the  spindle  is 
directed  dorsoventrally,  but  in  many  cases  the  spindle  may  be  tilted 
obliquely  in  an  anteroposterior  direction  (pi.  1,  fig.  12). 

The  fact  that  cysts  found  in  the  faeces  are  in  the  same  stage  of 
development  as  those  found  in  the  small  intestine  makes  it  appear 
that  further  development  of  these  cysts  in  the  intestine  is  arrested. 
The  two-zooid  somatella  present  in  the  binary  cysts  was  never  found 
to  have  undergone  plasmotomy  while  in  the  cyst,  as  was  the  case  in 
G.  microti  found  in  the  meadow  mouse.  It  is  very  probable  that 
further  development  is  dependent  on  the  cysts  being  ingested  by 
another  rat.  The  action  of  the  enzymes  of  the  stomach  it  is  believed 
serves  to  prepare  the  cyst  wall  for  digestion  when  the  cyst  reaches 
the  small  intestine;  otherwise,  we  should  expect  those  cysts  found  in 
the  small  intestine  before  the  defaecation  to  lose  their  walls,  without 
making  ejection  of  the  cysts  from  the  rat  and  subsequent  ingestion 
necessary.  The  reason  why  the  development  of  this  species  of  Giardia 
found  in  the  rat  should  be  arrested  during  encystment  is  a  matter  of 


118  University  of  California  Publications  in  Zoology        \yoi..  19 

conjecture  at  this  time.  It  is  very  probable  that  this  species  is  different 
from  G.  microti  although  their  structure  is  very  similar.  If  it  is  a  new 
species  then  it  is  very  probable  that  there  is  simply  a  difference  in  the 
rate  of  development  between  G.  microti  and  this  new  species.  The 
first  stages  of  development  are  similar  in  all  respects  and  there  is 
reason  to  believe  that  the  following  stages  are  also  similar.  The  greatest 
difference  is  in  the  location  of  the  different  stages  in  the  various 
regions  of  the  intestine.  The  fact  that  binary  cysts  were  found  in 
the  small  intestine  leads  one  to  believe  that  the  early  stages  of 
development,  including  the  formation  of  the  two-zooid  somatella  in 
this  new  species,  were  more  rapid  than  the  corresponding  rate  in  the 
formation  of  these  stages  in  G.  microti,  but  that  after  the  cysts  had 
gone  through  these  stages  of  binary  fission  the  rest  of  the  develop- 
ment was  slower  or  arrested  when  compared  with  G.  microti,  which 
continued  to  pass  through  all  the  other  stages  of  binary  fission  even 
to  the  completion  of  plasmotomy  within  the  cyst. 

On  the  other  hand,  the  presence  of  cysts  in  the  faeces  in  an  early 
stage  of  binary  fission  might  be  accounted  for  by  a  more  rapid  rate 
of  peristalsis  in  the  rat  compared  with  the  rate  of  peristalsis  in  the 
meadow  mouse.  In  order  to  test  this  hypothesis  the  cysts  which  were 
found  in  the  faeces  were  incubated  at  31°  C  for  several  days  contin- 
ously  and  examined  at  intervals  of  one,  four,  and  five  days.  The 
temperature  of  31°  G  was  chosen  because  it  was  lower  than  the 
optimum  of  most  of  the  bacteria  found  in  the  faeces.  This  would  tend 
to  prevent  excessive  proliferation  on  the  part  of  the  bacteria  which 
would  hasten  the  death  of  the  encysted  flagellates.  This  temperature 
is  considerably  lower  than  the  body  temperature  of  the  rat,  but  it  has 
been  used  for  the  cultivation  of  Protozoa  with  some  degree  of  success. 

Upon  the  examination  of  the  cysts  at  the  intervals  stated  above  no 
noticeable  changes  in  the  development  within  the  cysts  were  found. 
The  condition  of  the  body  within  the  cyst  was  identical  with  that  when 
the  cysts  were  defaecated.  The  cysts  decreased  markedly  in  their 
number,  which  may  be  due  to  their  death  through  the  agency  of  the 
bacteria.  The  incubation  failed  to  cause  any  further  development 
but  it  cannot  be  said  that  this  experiment  disproves  the  supposition 
of  the  effect  of  peristalsis.  Incubation  with  higher  temperatures  and 
with  cultures  of  cysts  free  from  bacteria  should  be  tried  before  con- 
demning the  hypothesis. 


1919]  Boeck:  Studies  on  Giardia  Microti  119 

Summary 

From  the  data  which  has  been  reviewed  in  the  foregoing  pages  the 
following  facts  regarding  the  life  cycle  of  G.  microti  are  established. 

There  are  two  distinct  phases  in  the  life  history  of  the  flagellate : 
the  one  a  free  vegetative  phase;  the  other  encystment. 

During  the  vegetative  phase  the  animal  may  pass  through  the 
following  stages: 

As  an  adult  organism  it  may  undergo  mitosis  in  which  case  there  is : 

Subsequent  formation  of  a  two-zooid  somatella  and  later  plas- 
motomy  to  form  two  daughter  flagellates;  or, 

The  formation  of  an  eight-zooid  somatella,  followed  by  plasmotomy 
to  form  eight  daughter  flagellates;  or, 

A  single  flagellate  may  encyst. 

During  encystment  the  animal  may  pass  through  the  following 
stages : 

By  binary  fission  two  daughter  flagellates  may  form  within  the  cyst 
which  will  be  liberated  when  the  cysts  are  ingested  by  another  host. 

By  multiple  fission  an  eight-zooid  somatella  may  be  formed  which 
will  be  liberated  to  complete  plasmotomy  upon  the  ingestion  of  the 
cyst  by  another  host  and  the  digestion  of  the  cyst  wall. 

In  case  cysts  should  be  ingested  before  they  have  completed  either 
binary  or  multiple  fission,  a  somatella  would  be  liberated  upon  the 
digestion  of  the  cyst  wall  which  would  continue  its  development  as  a 
somatella  in  a  free  state. 


120  University  of  California  Publications  in  Zoology       U^ou  19 

THE  PARABASAL  BODIES  OF  GIARDIA  MICROTI 

Introduction 

In  many  of  the  parasitic  Protozoa  the  process  of  nutrition  does 
not  always  result  in  a  growth  of  the  cytoplasmic  body,  but  instead 
there  may  result  the  formation  of  bodies  in  the  cytoplasm,  which  act 
as  reserve  food  materials.  These  bodies  may  be  intimately  connected 
with  the  motor  activity  of  the  organism,  especially  with  the  meta- 
bolic activity,  in  which  case  they  will  be  used  later  during  rapid 
growth,  during  reproductive  periods,  or  during  encystment,  when  the 
original  source  of  nutrition  is  cut  off. 

These  bodies  have  been  called  metaplastic  bodies  because  they  result 
from  the  metabolic  activity  of  the  organisms  and  are  deposited  in  the 
cytoplasm.  Among  these  bodies  may  be  cited  the  paramylum  grains 
of  flagellates,  the  paraglycogen  grains  of  gregarines  and  ciliates,  the 
plastinoid  granules  of  coccidia,  and  the  parabasal  bodies  of  flagellates. 

Function  of  the  Parabasal  Bodies 

In  a  previous  paper  by  the  author  (1917)  a  short  account  was 
'given  of  the  behavior  of  the  parabasal  bodies  of  Giardia  microti.  It 
was  pointed  out  then  that  these  organs  were  metabolic  reserve  centers 
of  food  materials,  which  acted  as  ''conveniences  on  the  part  of  the 
flagellate  for  coping  with  the  intestinal  medium  in  which  it  lives. 
They  appeared  to  be  more  intimately  connected  with  the  metabolic 
activity  of  the  parasite  rather  than  its  motor  activity. '' 

This  hypothesis  was  based  only  upon  morphological  aspects  of 
these  bodies  during  encystment  and  during  the  life  of  the  parasite  in 
the  free  state.  For  the  sake  of  completeness  the  activity  of  these 
parabasal  bodies  during  encystment  and  in  the  free  state  of  the  flagel- 
late may  be  reviewed. 

In  the  free-living  adult  (full-sized)  flagellate  there  are  two  para- 
basal bodies  located  in  the  posterior  third  of  the  body  and  lying  dorsal 
and  usually  across  the  axostyle.  The  bodies  are  usually  elongate  fusi- 
form in  shape.  In  the  cyst  the  bodies  may  be  two  or  more  in  number 
and  are  greatly  hypertrophied  at  the  beginning  of  encystment.  In 
the  cyst  the  bodies  are  located  in  various  regions  but  always  in  the 
posterior  part  of  the  body.     They  often  spread  out  like  the  tail  of  a 


1919]  Boeck:  Studies  on  Giardia  Microti  121 

comet  (pi.  1,  figs.  1,  4,  5,  13,  15,  16),  or  they  may  appear  as  conglo- 
merations of  a  material,  cloud-like  in  character  through  the  cytoplasm. 
Such  was  the  condition  of  the  parabasal  bodies  during  multiple  fission 
in  G.  muris  (Kofoid  and  Christiansen,  1915). 

During  mitosis  in  the  free  state  of  G.  microti  the  parabasal  bodies 
are  always  present  in  the  stages  of  the  prophase  and  in  the  metaphase, 
but  they  were  often  lacking  in  the  anaphase  and  telophase  and 
almost  without  exception  they  are  missing  in  the  stages  of  plasmotomy. 
The  only  exception  was  an  instance  described  in  which  a  parabasal 
body  was  seen  in  one  of  the  daughter  flagellates  in  the  process  of 
plasmotomy  of  a  two-zooid  somatella  in  the  free  state  (Boeck,  1917). 
In  the  investigations  upon  G.  muris  during  mitosis  the  parabasal 
bodies  were  absent  in  the  late  phases  of  mitosis  and  always  absent  in 
the  two-zooid  somatellas  resulting  from  binary  fission  (Kofoid  and 
Christiansen,  1915).  Likewise  in  the  four-zooid  somatella  resulting 
from  multiple  fission  of  a  flagellate  in  the  free  state,  the  parabasal 
bodies  were  lacking. 

With  the  encystment  of  G.  microti  the  parabasal  bodies  hyper- 
trophy to  a  great  extent  (pi.  1,  fig.  1)  and  as  the  process  of  binary 
fission  within  the  cyst  continues  the  bodies  disappear  (pi.  1,  figs.  6-8). 
In  multiple  fission  within  the  cyst  of  G.  microti,  there  also  occurs  an 
increase  in  size  of  the  parabasal  bodies  (pi.  1,  fig.  9),  and  when  the 
sixteen  nuclei  have  been  formed  in  many  cases  the  parabasal  bodies 
are  absent  or  they  appear  faint  (pi.  1,  fig.  11).  The  somatella  stages 
found  in  a  free  state  may  have  resulted  from  their  liberation  from  a 
cyst  by  the  digesting  away  of  the  cyst  wall,  and  in  these  stages,  it 
has  been  pointed  out,  the  parabasal  bodies  have  disappeared. 

It  was  on  these  morphological  aspects  of  the  parabasal  bodies  dur- 
ing mitosis  and  during  encystment  that  the  conclusion  was  reached 
that  these  bodies  were  reserve  food  centers  to  be  utilized  during  periods 
of  reproduction  and  during  encystment,  when  the  original  source  of 
food  supply  had  been  cut  off.  At  such  periods  as  these  there  is  an 
extra  drain  on  the  food  supply  of  the  flagellate,  since  the  rate  of 
metabolism  during  reproductive  activity  is  great,  and  since  during 
encystment  not  only  are  reproductive  processes  carried  on,  necessitating 
a  drain  on  the  food  supply,  but  also  encystment  itself  may  extend  over 
a  long  period  of  time,  which  necessitates  an  extra  food  supply.  This 
food  reserve  is  depleted  at  the  end  of  encystment,  when,  for  the  most 
part,  the  reproductive  processes  within  have  also  been  completed. 


122  University  of  California  Publications  in  Zoology        [yoL.  19 

Biochemical  and  Staining  Qualities  op  the  Parabasal  Bodies 

More  evidence,  not  morphological  but  biochemical  in  nature,  is 
required  to  determine  the  function  of  the  parabasal  bodies. 

When  either  the  free  forms  or  cysts  of  G.  microti  are  stained  with 
Heidenhain's  iron  haemotoxylin  the  parabasal  bodies  appear  very 
dark,  and  for  this  reason  it  was  previously  thought  that  these  bodies 
were  chromatoidal  in  nature  and  of  probable  chromatinic  origin.  In 
Giardia  microti,  however,  there  has  been  no  evidence  of  chromidia 
within  the  nuclei  or  escaping  from  the  nuclei.  Again,  increase  in  size 
of  the  parabasal  bodies  is  not  accompanied  by  a  decrease  in  size  of  the 
karyosome  of  the  nuclei,  so  this  evidence  would  militate  against  the 
origin  of  the  parabasal  bodies  from  the  nuclei. 

Since  iron  haemotoxylin  is  a  selective  stain  for  both  chromatin 
and  cytoplasmic  structures,  which  in  most  cases  are  of  cytoplasmic 
origin,  it  was  thought  more  prudent  to  use  other  stains  substantively, 
in  order  to  fix  the  chromatophilly  of  the  parabasal  bodies.  When  the 
preparations  of  flagellates  and  cysts  were  stained  with  acid  fuchsin 
and  methyl  green  the  parabasal  bodies  appeared  red  and  the  chromatin 
of  the  nuclei  appeared  green.  From  this  reaction  the  parabasal  bodies 
were  thought  to  be  acidophillic,  and  so  differed  from  the  chromatin 
of  the  nuclei  which  was  basophyllic.  In  about  half  of  the  flagellates 
the  parabasal  bodies  appeared  to  be  situated  upon  or  in  a  definite 
area  of  the  cytoplasm,  limited  by  a  membrane-like  structure.  In 
other  cases  the  two  parabasal  bodies  appeared  as  if  stained  by  iron 
haemotoxylin,  i.e.,  just  two  fusiform  bodies.  But  when  the  area 
enclosed  by  a  membrane-like  structure  appeared,  that  portion  of  the 
area  not  occupied  directly  by  the  parabasal  bodies  was  lighter  in 
staining  reaction  than  the  parabasal  bodies  or  the  cytoplasm  surround- 
ing this  parabasal  area.  There  seemed  to  be  evidence  of  another 
substance  along  with  the  parabasal  bodies,  the  two  together  constituting 
the  parabasal  complex,  so  to  speak.  This  other  substance  might  well 
be  the  ground  substance,  or  parabasal-plasm  within  which  the  para- 
basal bodies  are  situated. 

To  substantiate  the  acidophyllic  nature  of  the  parabasal  bodies 
preparations  were  treated  with  basic  fuchsin.  In  these  preparations 
no  flagellate  was  seen  in  which  the  parabasal  bodies  or  parabasal- 
plasm  was  stained,  but  in  all  cases  the  parabasal-plasmic  region  was 
identified  as  a  more  refractive  area  than  the  surrounding  cytoplasm. 
It  was  certain,  then,  that  the  constitution  of  the  parabasal  bodies 


1919]  Boeck:  Studies  on  Giardia  Microti  123 

differed  from  that  of  the  chromatin  of  the  nuclei,  that  their  consti- 
tution was  aeidophyllic.  Some  of  the  very  preparations  which  when 
stained  with  basic  fuchsin  revealed  no  parabasals  were  subsequently 
treated  with  iron  haematoxylin  and  the  parabasals  reappeared ;  they 
had  been  present  but  the  basic  fuchsin  did  not  stain  them. 

Alexeieff's  (1917)  work  with  flagellates,  especially  Trichomonas 
aiigusta,  has  shown  that  the  parabasal  rod  is  of  mitochondrial  con- 
stitution. Instead  of  the  rod,  in  times  of  division  there  may  be  a  row 
of  mitochondrial  granules  which  he  believes  form  the  parabasal  rod 
by  forming  chondriomites.  He  has  also  shown  that  the  mitochondria 
are  structures  which  secrete  glycogen,  which  is  utilized  by  the  motor 
and  metabolic  activity  of  the  organism. 

When  preparations  of  free  flagellates  killed  in  Schaudinn's  fluid 
and  fixed  in  ninety-five  per  cent  alcohol  were  treated  with  Lugol's 
solution  to  test  for  glycogen  in  the  parabasal  bodies  two  distinct 
conditions  of  the  parabasal-plasm  w^ere  noted.  The  parabasal  bodies 
themselves  did  not  appear  but  only  the  glycogen  contained  in  the 
parabasal-plasm.  In  the  first  place  the  glycogen  was  seen  as  a  rec- 
tangular, mahogany  colored  mass  filling  all  the  parabasal-plasm  for  no 
membrane-like  structure  could  be  detected ;  or  the  glycogen  assumed  a 
thin  rod-shaped  mass,  or  was  in  two  smaller  masses.  In  the  latter  two 
cases,  however,  these  glycogen  masses  were  lying  the  parabasal-plasm, 
which  was  easily  identified  by  the  more  darkly  stained,  ovoid,  mem- 
brane-like structure  which  surrounded  them.  In  the  second  place,  other 
individuals  did  not  show  any  glycogen  present  in  the  parabasal-plasm, 
but  the  area  itself  was  clearly  seen  as  a  refractive,  rectangular  body 
lying  in  a  position  identical  with  that  occupied  by  the  parabasal  bodies. 
The  cases  which  revealed  no  glycogen  in  the  parabasal-plasm  were 
about  the  same  in  number  as  those  that  showed  glycogen  present.  Out 
of  fifty  flagellates,  twenty-three  showed  glycogen  present,  twenty-four 
showed  the  absence  of  the  glycogen,  and  three  flagellates  showed  no 
glycogen  or  parabasal-plasm  present.  These  three  casese  Avere  inter- 
preted to  mean  the  complete  absence  of  the  parabasal  bodies  and 
parabasal-plasm. 

It  was  thought  at  the  time  that  perhaps  those  flagellates  which 
showed  only  the  parabasal-plasm  present  actually  lacked  parabasal 
bodies.  Accordingly  some  of  the  preparations  treated  with  Lugol's 
solution  were  stained  with  iron  haemotoxylin,  and  after  examining  fifty 
flagellates  forty-seven  were  found  to  have  parabasal  bodies  and  three 
did  not.    This  was  identical  with  the  ratio  counted  in  the  preparation 


124  University  of  California  Fublications  in  Zoology        [^ol.  19 

treated  with  only  Lugol's  solution.  But  out  of  the  forty-seven  flagel- 
lates showing  parabasal  bodies  present  twenty  of  them  showed  the 
presence  of  the  parabasal-plasm.  lying  between  the  parabasal  bodies. 
This  was  attributed  to  previous  treatment  with  iodine  since  prep- 
arations not  treated  with  iodine  previous  to  staining  with  iron  haema- 
toxylin,  fail  to  show  as  distinctly  the  presence  of  the  parabasal-plasm. 
Some  glycogen  is  lost  through  processes  of  killing  and  fixation  of 
the  flagellates,  so  that  the  glycogen  which  remains  is  only  a  portion  of 
the  original  amount  present  in  the  parabasal-plasm.  In  view  of  this 
fact,  in  the  case  of  those  flagellates  which  showed  only  parabasal-plasm 
without  glycogen,  the  lack  of  glycogen  may  have  been  due  to  the  loss 
of  the  comparatively  small  amount  present  in  the  parabasal-plasm 
previous  to  the  killing  and  fixation  of  the  preparations. 

Origin  of  Parabasal  Bodies 

The  origin  of  the  mitochondrial  granules  found  in  Trichomonas 
augusta  around  the  nucleus,  along  the  parabasal  rod,  and  in  the  axo- 
style  may  be  of  nuclear  origin  since  previous  to  mitotic  activity  a 
distinct  chromidial  cloud  is  seen  about  the  nucleus.  These  chromidia 
may  have  been  extruded  from  the  nucleus  since  there  is  also  an 
intranuclear  chromidial  cloud  present  at  the  same  time.  Alexeieff 
(1917)  also  finds  that  the  mitochondrial  granules  are  azurophyllic 
when  stained  with  Giemsa.  This  reaction  being  characteristic  of  the 
chromatin  of  the  nucleus  Alexeieff  believes  that  the  reaction  is  further 
evidence  of  the  probable  nuclear  origin  of  the  granules.  It  seems 
safe  to  infer  from  this  paper  that  if  the  mitochondrial  granules  form 
the  parabasal  rod  indirectly  this  rod  is  also  of  nuclear  origin. 

It  has  been  mentioned  previously,  however,  that  in  G.  microti  there 
is  no  evidence  of  chromidial  extrusion  or  chromidial  clouds,  and 
because  the  parabasal  bodies  are  markedly  acidophyllic  there  seems 
little  evidence  for  attributing  nuclear  origin  to  these  organs.  Again, 
glacial  acetic  acid  in  the  killing  fluid  failed  to  dissolve  the  parabasal 
bodies,  which  would  have  been  expected  if  they  were  mitochondrial 
in  constitution.  From  the  evidence  at  hand  it  appears  best  to  desig- 
nate these  parabasal  bodies  along  with  the  parabasal-plasm  as  struc- 
tures derived  directly  from  the  cytoplasm.  They  are  metaplastic  in 
nature,  since  they  are  formed  from  the  anabolic  processes  of  metabolism 
and  tend  to  disappear  when  the  katabolic  activity  exceeds  anabolism. 
This  is  especially  marked  during  the  final  stages  of  mitosis,  plasmotomy 


1919]  Boeck:  Studies  on  Giardia  Microti  125 

and  encystment  with  its  incident  processes  of  reproduction.  When 
the  parabasal  bodies  have  disappeared  they  are  reformed  from  sub- 
stances out  of  the  cytoplasm,  when  metabolism  is  again  normal. 

Alexeieff  (1917)  has  termed  the  parabasal  bodies  of  flagellates 
the  ' '  kinetoplaste ; ' '  but  it  does  not  seem  that  this  name  is  appropriate 
for  the  parabasal  organs  of  Giardia  since  as  we  have  seen  that  there  are 
actually  two  substances  which  make  up  the  parabasal  complex.  In 
cognizance  of  the  presence  of  the  parabasal  bodies  themselves  lying 
in  or  upon  another  substance  it  seems  to  the  author  best  to  refer  to 
the  bodies  as  parabasal  bodies  which  secrete  glycogen.  The  glycogen 
is  stored  up  in  the  other  substance,  the  ground  work  or  paratasal- 
plasm,.  The  word  kinetoplaste  connotes  the  motor  but  not  the  metabolic 
significance  of  the  parabasal  bodies  and  because,  as  we  have  seen, 
the  parabasal  bodies  are  more  intimately  correlated  with  the  metabolic 
activity  of  Giardia  it  is  best  to  discard  the  word  and  to  still  refer  to 
the  complex  as  parabasal  bodies  and  parabasal-plasm. 

Summary 

From  the  foregoing  data  the  parabasal  bodies  were  found  to  be : 

Acidophyllic  in  constitution. 

They  are  of  cytoplasm  origin. 

Their  function  is  to  secrete  glycogen  which  is  retained  for  sub- 
sequent use  in  the  parabasal-plasm.  The  glycogen  constitutes  a  re- 
serve food  supply  which  is  utilized  during  the  reproductive  period 
and  during  encystment. 


126  University  of  California  Publications  in  Zoology       [Vol.  19 


THE    THERAPEUTIC   VALUE    OF   BISMUTH   SUBNITRATE 

AND  BISMUTH  SALICYLATE  IN  THE  TREATMENT 

OP  GIARDIASIS  (LAMBLIASIS)  OF  RATS 

Introduction 

The  use  of  these  two  salts  in  the  chemotherapy  of  giardiasis  of 
man  was  attended  with  some  success  in  England  and  consequently 
encouraged  further  treatments  with  these  same  chemical  compounds 
in  order  to  ascertain  their  true  therapeutic  value. 

The  account  of  the  bismuth  treatments  of  giardiasis  of  man  was 
given  by  Porter  (1916),  who  made  an  enumerative  study  of  the  cysts 
of  Giardia  intestinalis  occurring  in  the  stools  of  dysenteric  patients. 

Procedure 

Finding  a  large  number  of  rats  infected  with  G,  microti,  a  species 
similar  to  G.  intestinalis  (the  history  of  which  was  followed  for  a 
period  of  one  month  by  daily  faecal  examinations)  six  were  selected 
which  showed  the  heaviest  infection.  The  degree  of  infection  was 
determined  by  the  number  of  positive  examinations  of  the  faeces  made 
daily  during  the  month.  Three  of  the  rats  were  treated  with  bismuth 
sub-nitrate  and  the  other  three  with  bismuth  salicylate. 

It  was  found  that  the  best  way  to  administer  the  dose  is  to  spread 
the  salt  (powder)  on  water-soaked  bread  each  day.  The  rats  then  ate 
the  bread  at  the  same  time,  receiving  approximately  a  full  dose  of  the 
salt.  The  rats  did  not  object  to  the  treatment,  although  at  different 
intervals  they  appeared  very  nervous,  slow  in  movement,  often  sluggish, 
to  some  degree  ferocious,  and  their  coats  manifested  a  certain  degree 
of  roughness.  Periods  of  constipation  also  occurred,  and  in  the 
case  of  one  rat  no  faeces  were  defaecated  on  one  day.  Otherwise 
constipation  was  indicated  by  the  defaecation  of  very  small  pellets. 

As  has  been  said  previously,  a  history  of  the  six  rats  was  known 
throughout  a  period  of  about  a  month.  Table  4  shows  the  degree  of 
infection  of  each  rat  for  each  day  during  a  period  of  twenty-eight 
days  when  daily  examinations  had  been  made  of  the  faeces.  The 
number  in  each  square  represents  the  number  of  cysts  counted  for  that 
day  in  any  twenty  fields  of  the  microscope  by  the  use  of  a  one-inch 
ocular  and  four-millimeter  objective.  A  negative  sign  in  the  square 
means  that  no  cysts  were  detected  in  the  stools  for  that  day. 


1919]  Boeck:  Studies  on  Giardia  Microti  127 


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128  V Oliver sity  of  California  Publications  in  Zoology        \yoi..  19 

The  treatments  began  on  December  18,  1917,  and  continued  for 
three  weeks,  until  January  8,  1918.  The  dose  for  man  was  a  tea- 
spoonful  of  either  of  the  bismuth  salts  three  times  a  day,  and  usually 
after  five  to  ten  days  of  treatments  the  infection  appeared  to  be  cured 
(Porter,  1916).  This  dose  for  man  amounted  to  about  3.6  to  4  gram? 
of  the  chemicals  each  day. 

To  arrive  at  dosage  for  the  rats  a  dose  was  proportioned  accord- 
ing to  the  ratio  of  the  body  weight  of  man  (about  146  pounds)  and 
the  average  weight  of  the  rats  (about  300  grams).  This  dose  was 
found  to  be  about  20  milligrams  each  day.  This  was  the  first  dose  tried 
with  the  rats,  but  was  considerably  increased  as  the  treatments  pro- 
gressed (table  5). 

In  making  the  daily  faecal  examinations  of  these  six  rats  faecal 
emulsions  of  the  stools  were  prepared  in  distilled  water  each  day 

during  the  course  of  treatment.    A  drop  of  this  emulsion  was  trans- 

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ferred  to  a  slide,  adding  to  it  a  drop  of  neutral  red  solution  ^ 

(Boeck,  1917),  and  then  the  preparation  was  examined  for  the  cysts 
of  Giardia. 

When  the  cysts  were  found  their  number  was  counted  in  any 
twenty  fields  of  the  microscope,  using  a  one-inch  ocular  and  a  four- 
millimeter  objective.  A  plus  sign  was  placed  in  the  square  of  the 
table  (table  5)  for  that  day  and  the  number  of  cysts  counted  was 
placed  above  the  sign.  If  no  cysts  were  detected  in  the  first  cover- 
slip  preparation  from  the  emulsion,  after  stirring  the  emulsion  a 
second  preparation  was  made  and  by  the  same  procedure  a  third 
preparation  if  the  second  one  turned  out  to  be  negative.  At  the  end 
of  three  preparations  no  cysts  were  detected,  then  the  stools  were 
designated  negative  for  that  day,  and  a  minus  sign  was  written  in 
the  square  of  the  table  for  that  day.  If,  however,  cysts  were  found 
in  either  the  second  or  third  preparation  their  number  was  counted 
and  a  plus  sign  written  in  the  square  for  that  day,  placing  the  number 
of  cysts  counted  above  the  sign  and  the  number  of  the  preparation 
below  the  sign.  It  took  three  negative  examinations  to  make  a  rat 
negative  for  that  day  on  which  the  stools  were  collected. 

The  dose  of  twenty  milligrams  was  first  administered  to  the  rats. 
The  dose  was  the  same  for  the  bismuth  subnitrate  and  bismuth  sali- 
cylate. After  seven  treatments  with  a  dose  of  twenty  milligrams  the 
daily  examinations  still  showed  rats  1,  4,  9,  and  12  to  be  infected,  but 
rats  8  and  10  showed  no  evidence  of  infection,  there  being  no  cysts  in 


1919]  Boeck:  Studies  on  Giardia  Microti  129 


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130  University  of  California  Publications  in  Zoology       [Vol.  19 

the  faeces.  On  December  25  the  dose  was  increased  to  thirty  milli- 
grams for  two  days  and  then  to  fifty  milligrams  for  three  days,  for 
the  fact  that  four  rats  remained  infected  led  one  to  believe  that  the 
dose  previously  used  in  the  treatments  was  insufficient  to  effect  a  cure. 

After  increasing  the  dose  to  fifty  milligrams  the  crystals  of  the 
bismuth  subnitrate  were  seen  in  the  faeces  of  rats  4,  9,  and  10.  The 
dose  certainly  must  have  been  adequate  since  the  faeces  contained  a 
great  amount  of  the  crystals,  and  the  chemicals  must  have  come  in 
contact  with  the  flagellates. 

The  rats  appeared  quite  ill  as  a  result  of  the  large  dose  and  the 
treatment  was  dispensed  with  for  two  days.  On  January  1  a  dose  of 
thirty  milligrams  was  again  administered  and  this  dose  was  increased 
to  sixty  milligrams  on  the  following  day.  A  dose  of  thirty  milligrams 
followed  by  fifty  milligrams  was  given  the  rats  on  the  next  two  suc- 
cessive days.  Treatment  was  suspended  on  January  5  and  6,  and 
sixty  milligrams  were  given  on  the  last  day. 

At  the  end  of  this  treatment  with  these  two  bismuth  salts  rats 
1,  4,  9,  and  12  were  still  infected.  Cysts  were  found  in  the  faeces  of 
all  these  rats,  except  rat  4  on  the  last  day.  But  rat  4  showed  cysts 
in  the  faeces  two  days  before  the  treatments  were  concluded.  Only 
rat  8,  treated  with  bismuth  salicylate,  and  rat  10,  treated  with  bismuth 
subnitrate,  appeared  to  be  cured. 


Discussion 

From  table  4  we  see  that  these  two  rats,  8  and  10,  were  heavily 
infected  during  the  twenty-eight  days  that  their  faeces  were  examined. 
But  during  the  treatment  cysts  were  found  in  the  faeces  of  rat  8 
on  only  two  different  and  successive  days,  while  cysts  were  found  in 
the  faeces  of  rat  10  only  on  one  day.  Apparently  the  drug  had  cured 
these  rats. 

The  rats  were  then  posted  and  at  autopsy  no  sign  of  pathological 
disturbance  of  the  organs  was  seen.  The  jejunum,  the  seat  of  infec- 
tion by  Giardia  in  rats,  was  not  discolored  nor  did  it  contain  any  gas. 
In  cases  of  infection  the  jejunum  is  usually  orange  colored  and  con- 
tains gas.  The  upper  and  lower  duodenum,  jejunum,  and  ileum  were 
examined  for  free  forms  and  cysts  of  Giardia  and  none  were  found,  nor 
were  there  any  cysts  in  the  colon  or  rectum.  Oct  omit  us  muris  was 
very  abundant  in  the  jejunum  and  ileum.     The  absence  of  Giardia 


1919]  Boeck:  Studies  on  Giardia  Microti  131 

in  both  these  rats  at  autopsy,  showed  them  to  be  free  from  infection  by 
this  flagellate. 

It  would  seem  that  these  two  rats,  8  and  10,  were  cured  by  the  action 
of  the  chemicals,  but  such  a  conclusion  is  not  altogether  warranted 
when  we  consider  that  the  other  four  rats  remained  infected.  Another 
interpretation  of  these  two  cases  is  justifiable,  viz. :  In  the  study  of  the 
cycle  of  encystment  in  G.  microti  rat  3  showed  only  one  period  when 
the  cysts  were  found  in  the  faeces.  At  autopsy  this  rat  proved  to  be 
free  from  an  infection  by  Giardia  and  the  conclusion  reached  was  that 
the  rat  had  been  capable  of  ridding  itself  of  the  infection,  one  way  or 
another. 

It  will  be  noticed  that  in  the  cases  of  rats  8  and  10,  w^hich  appeared 
cured  by  the  chemicals,  treatment  with  these  salts  of  bismuth  did  not 
take  place  until  December  18.  This  date  was  twenty-six  days,  approxi- 
mately four  weeks,  after  the  day  when  their  period  of  infection  of 
twenty-eight  days,  November  21,  had  been  concluded.  There  was  an 
interim,  then,  of  nearly  four  weeks  during  which  these  two  rats  could 
have  thrown  off  the  infection. 

If  rats  8  and  10  threw  off  the  infection  previous  to  the  treatment 
with  the  salts  of  bismuth  then  all  the  days  subsequent  should  have 
showed  no  cysts  in  the  faeces.  This,  however,  was  not  the  case  for 
rat  8  showed  cysts  in  the  faeces  on  two  successive  days,  December  27 
and  28,  and  rat  10  showed  cysts  in  the  faeces  on  December  22.  A 
possible  explanation  of  the  presence  of  these  cysts  may  be  that  they 
are  perhaps  the  result  of  a  short  period  of  reinfection. 

Reinfection  might  have  occurred  by  the  transfer  of  pellets  from 
rat  9  into  the  cages  of  rats  8  and  10.  Rat  9  escaped  from  its  cage 
one  night  and  was  found  the  next  day  running  along  the  cages  of 
the  other  rats.  If  reinfection  took  place  then,  according  to  the  data 
both  rats  8  and  10  must  have  thrown  off  the  infection  for  the  second 
time  for  no  cysts  were  in  the  faeces  after  the  single  period  of  infection 
in  either  of  the  rats  and  they  were  not  present  at  autopsy. 

Even  though  reinfection  might  have  taken  place  it  is  altogether 
probable  that  the  salts  of  bismuth  did  cure  these  two  rats;  but  the 
persistence  of  the  infection  in  the  other  four  rats  certainly  militates 
against  the  practicability  of  these  chemicals  as  a  specific  cure  for 
giardiasis. 

Porter  (1916)  reports  the  cases  of  three  men  infected  with  G.  intes- 
tinalis  and  treated  with  bismuth  salts  in  which  an  apparent  cure  was 
affected.     It  is  significant  to  note  that  daily  faecal  examinations  of 


132  University  of  California  Publications  in  Zoology        ['Voj..  19 

these  men  were  not  made  after  the  time  the  cysts  disappeared  from 
the  stools,  following  treatment  with  the  chemicals.  These  men  were 
then  discharged  from  the  hospital  as  cured  from  giardiasis.  Porter 
recognized  the  presence  of  a  cycle  of  encystmtent  in  G.  intestinalis,  but 
this  factor  was  not  taken  into  account  when  the  men  were  determined 
free  from  infection.  It* is  very  probable  if  these  men  who  appeared 
cured  could  have  been  watched  for  several  days  longer  and  their 
stools  examined  daily,  that  the  cysts  might  have  recurred.  The  fact 
that  the  cysts  were  absent  from  the  stools  after  several  treatments 
with  the  salts  of  bismuth  was  not  conclusive  proof  that  the  men  were 
free  from  the  infection  of  Giardia;  this  could  only  have  been  ascer- 
tained by  a  series  of  daily  examinations  for  the  purpose  of  determining 
whether  or  not  the  cysts  recurred  in  the  faeces  at  the  proper  time 
in  the  cycle. 

Summary 

From  the  experimental  work  conducted  with  the  salts  of  bismuth 
on  rats  infected  with  Giardia  and  the  results  obtained  with  the 
treatment  of  giardiasis  in  man  with  the  same  chemicals  it  is  certain 
that  the  therapeutic  value  of  bismuth  subnitrate  and  bismuth  salicylate 
is  negative  in  the  treatment  of  giardiasis.  The  conclusion  is  supported 
by  W.  L.  Yakimoff,  W.  J.  Wassilevski,  and  N.  A.  Zwietkoff  (1918), 
who  state  the  inefficacy  of  these  chemicals  in  the  chemotherapy  of  this 
disease. 


1919]  Boeck:  Studies  on  Giardia  Microti  133 


LITERATURE  CITED 

Alexeieff,  a. 

1917a.  Nature  mitochondriale  du  corps  parabasal  des  flagelles.     C.-E.,  Soc, 

Biol.  Paris,  80,  499-502,   1  fig.  in  text. 
1917&.  Sur  la  fonction  glycoplastique  du  kinetoplaste   (kinetonucleus)   chez 

les  Flagelles.     Ibid.,  80,  512-514. 
1917c.  Sur  les  mitochondries  a  fonction  glycoplastique.     Ibid.,  80,  510-512. 
Boeck,  W.  C. 

1917o.  Mitosis  in  Giardia  microti.    Univ.  Calif.  Publ.  Zool.,  18,  1-26,  1  plate, 

15  figs,  in  text. 
1917Z>.  A  rapid  method  for  the  detection  of  protozoan  cysts  in  mammalian 
faeces.     Ibid.,  18,  145-149. 
Calkins,  G.  N. 

1904.  Studies  on  the  life  history  of  Protozoa.     IV,  Death  of  the  A  series, 

Jour.  Exper.  Zool.,  1,  423-462,  3  pis.,  3  figs,  in  text. 
Cropper,  J.  W.  and  Row  R.  W.  H. 

1917.     A  method  of  concentrating  Entamoeba  cysts  in  stools.     Lancet,  192, 
179-182. 
Gregory,  L.  H. 

1909.     Observations    on    the   life   history    of    TilUna   magna.      Jour.   Exper. 
Zool.,  6,  383-432,  2  pis. 
KoFoiD,  C.  A.,  AND  Christiansen,  E.  B. 

1915.  On   binary    and    multiple   fission   in    Giardia  muris    (Grassi).      Univ. 

Calif.  Publ.  Zool.,  16,  30-54,  pis.  5-8,  1  fig.  in  text. 

KOFOID,  C.   A.,  AND   SWEZY,   O. 

1916.  Mitosis  and  multiple  fission  in  trichomonad  flagellates.     Proc.  Amer. 

Acad.  Arts  and  Sci.,  Boston,  51,  289-378,  pis.  1-8,  7  figs,  in  text. 
Porter,  A. 

1916.  An  enumerative  study  of  the  cysts  of  Giardia  (Lamblia)  intestinalis 

in  human  dysenteric  faeces.     Lancet,  190,  1166-1169,  7  charts. 

SCHAUDINN,  F. 

1903.     Untersuchungen   iiber   die  Fortpflanzung  einiger   Rhizopoden.     Arb. 
kais.  Gesundh.,  19,  547-576. 
Woodruff,  L.  L.,  and  Baitsell,  G.  A. 

1905.  Rhythms   in   the   reproductive    activity   of   Infusoria.     Jour.   Exper. 

Zool.,  11,  339-359,  13  figs,  in  text. 
1917.     The  Influence  of  general  environmental  conditions  on  the  periodicity  of 

endomixis  in  Paramecium  aurelia.     Biol.  Bull.  33,  437-462,  12  figs. 

in  text. 
Yakimoff,  W.  L.,  Wassilevski,  W.  J.,  and  Zwietkoff,  N.  A. 

1917.  Sur  la  chimiotherapie  de  la  lambliose.     C.  R.  Soc.  Biol.,  Paris,  80, 

506-507,  1  table. 


EXPLANATION  OF  PLATE 

All  figures  are  of  cysts  of  Giardia  microti  from  the  meadow  mouse  and  of  the 
cysts  of  Giardia  found  in  the  culture  rats,  drawn  with  camera  lucida  from  smear 
preparations.     Magnification,  X  2750. 

PLATE    1 

Fig.  1.  Ventral  view,  single-individual  cysts  found  in  small  intestine  and 
caecum;  nuclei  in  resting  stage.     Two  parabasals  dividing. 

Fig.  2.  Dorsal  view,  single-individual  cyst  found  in  caecum;  nuclei  divid- 
ing, parabasals  in  division. 

Fig.  3.  Dorsal  view,  single-individual  cyst  found  in  caecum  of  meadow 
mouse;  nuclei  in  anaphase,  small  parabasals,  axostyle  completely  split. 

Fig.  4.  Dorsal  or  ventral  view,  single-individual  cyst,  chromatin  in  one 
nucleus  divided  into  two  masses.  Parabasals  dividing.  Cyst  from  caecum 
of  meadow  mouse. 

Fig.  5.  Dorsoventral  view,  binary  cyst  from  colon  of  meadow  mouse; 
nuclei  divided,  two  parabasals. 

Fig.  6.  Dorsoventral  view,  binary  cyst  from  colon  of  meadow  mouse;  four 
nuclei,  new  organelles,  no  parabasals. 

Fig.  7.  Side  view,  binary  cyst  from  colon  of  meadow  mouse;  shows  plan 
of  cleavage  of  the  parent  body  to  form  two  daughter  flagellates.     No  parabasals. 

Fig.  8.  Side  view,  binary  cyst  from  colon  and  rectum  of  meadow  mouse. 
Two  complete  individuals  with  their  organelles,  no  parabasals. 

Fig.  9.  Dorsoventral  view,  multinucleate  cyst,  two  axostyles,  large  para- 
basals, twelve  nuclei.     Found  in  small  intestine  of  meadow  mouse. 

Fig.  10.  Dorsoventral  view,  multinucleate  cyst,  two  axostyles,  twelve 
nuclei,  parabasals.    Found  in  small  intestine  of  meadow  mouse. 

Fig.  11.  Dorsoventral  view,  multinucleate  cyst  from  small  intestine  of 
meadow  mouse;   two  axostyles,  no  parabasals,   sixteen  nuclei. 

Fig.  12.  Dorsal  view,  single-individual  cyst  from  jejunum  of  rat;  nuclei  in 
anaphase,  large  parabasals,  axostyle  partially  split. 

Fig.  13.  Dorsoventral  view,  binary  cyst  from  jejunum  of  rat;  four  nuclei, 
large  parabasals. 

Fig.  14.  Dorsoventral  view,  binary  cyst  from  colon  of  rat;  four  nuclei  and 
three  large  parabasals. 

Fig.  15.  Ventral  view,  binary  cyst  from  jejunum  of  rat;  four  nuclei  in 
resting  stage,  parabasals. 

Fig.  16.  Side  view,  binary  cyst  from  jejunum  of  rat;  four  nuclei  two  exo- 
styles,  large  parabasals. 

Many  of  the  cysts  in  the  preparations  show  a  shrinkage  away  from  the  cyst 
wall;  this  is  attributed  to  plasmolysis  at  the  time  of  fixation. 


[134] 


UNIV.    CALIF.    PUBL,    ZOOL,    VOL.    19 


[  BOECK  ]    PLATE    1 


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16 


UNIVEESITT  OF  CALirOElOA  PUBLICATIONS— (Continued) 

8.  Osteological  Relationships  of  Tliree  Species  of  Beavers,  by  F.  Harvey 

Holden.    Pp.  75-114,  plates  5-12,  18  text  figures.    Marcli,  1917 40 

9.  Notes  on  the  Systematic  Status  of  the  Toads  and  Frogs  of  California,  by 

Charles  Lewis  Camp.    Pp.  115-125,  3  text  figures.    February,  1917 10 

10.  A  Distributional  List  of  the  Amphibians  and  Eeptiles  of  California,  by 

Joseph  Grinnell  and  Charles  Lewis  Camp.    Pp.  127-208,  14  figures  in  text. 
July,  1917  - . ^      .85 

11.  A  Study  of  the  Races  of  the  White-Fronted  Goose  (Anser  aXbifrons)  Occur- 

ring in  California,  by  H.  S.  Swarth  and  Harold  O.  Bryant.    Pp.  209-222, 

2  figures  in  text,  plate  13.    October,  1917 ^      J.5 

12.  A  Synopsis  of  the  Bats  of  CaUf  omia,  by  Hilda  Wood  Grinnell.    Pp.  223-404, 

plates  14-24,  24  text  figures.    January  31,  1918  2.00 

13.  The  Pacific  Coast  Jays  of  the  Genus  Aphelocoma,  by  H.  S.  Swarth.    Pp. 

405-422,  1  figure  in  text.    February  23,  1918  . .20 

14.  Six  New  Mammals  from  the  Mohave  Desert  and  Inyo  Regions  of  California, 

by  Joseph  Grinnell.    Pp.  423-430. 

15.  Notes  on  Some  Bats  from  Alaska  and  British  Columbia,  by  Hilda  Wood 

GrinneU.    Pp.  431-433. 

Nos.  14  and  15  in  one  cover.    April,  1918 15 

16.  Revision  of  the  Rodent  Germs  Aplodontia,  by  Walter  P.  Taylor.    Pp.  435- 

504,  plates  25-29,  16  text  figures.    May,  1918 .75 

17.  The  Subspecies  of  the  Mountain  Chickadee,  by  Joseph  Grinnell.    Pp.  505- 

515,  3  text  figures.    May,  1918 .15 

18.  Excavations  of  Burrows  of  the  Rodent  Aplodontia,  with  Observations  on 

the  Habits  of  the  Animal,  by  Charles  Lewis  Camp.    Pp.  517-536,  6  figures 

in  text.    June,  1918  20 

Index,  pp.  537-545. 

Vol.  18.  1.  Mitosis  in  Giardia  microti,  by  William  C.  Boeck.  Pp.  1-26,  plate  1.  Octo- 
ber, 1917 ...::.^ , .36 

2.  An  Unusual  Extension  of  the  Distribution  of  the  Shipworm  in  San  Fran- 
cisco Bay,  California,  by  Albert  L.  Barrows.    Pp.  27-43.    December,  1917.      .20 

8.  Description  of  Some  New  Species  of  Polynoidae  from  the  Coast  of  Cali- 
fornia, by  Christine  Essenberg.    Pp.  45-60,  plates  2-3.    October,  1917 20 

4.  New  Species  of  Amphinomidae  from  the  Pacific  Coast,  by  Christine  Essen- 

berg.   Pp.  61-74,  plates  4-5.    October,  1917  15 

5.  Crithidia  euryophthalmi,  sp.  nov.,  from  the  Hemipteran  Bug,  Euryophthalmus 

convivus  St&l,  by  Irene  McCulloch.    Pp.  75-88,  35  text  figures.    Decem- 
ber, 1917 - .15 

6.  On  the  Orientation  of  Erythropsis,  by  Charles  Atwood  Kofoid  and  Olive 

Swezy.  Pp.  89-102,  12  figures  in  text.    December,  1917 15 

7.  The  Transmission  of  Nervous  Impulses  in  Relation  to  Locomotion  in  the 

Earthworm,  by  John  F.  Bovard.    Pp.  103-134,  14  figures  in  text.    January, 
1918 _ : 35 

8.  The  Function  of  the  Giant  Fibers  In  Earthworms,  by  John  F.  Bovard.    Pp. 

135-144,  1  figure  in  text.    January,  1918 10 

9.  A  Rapid  Method  for  the  Detection  of  Protozoan  Cysts  In  Mammalian 

Faeces,  by  William  C.  Boeck.    Pp.  145-149.    December,  1917 .05 

10.  The  Musculature  of  Heptanchus  maoulatus,  by  Pirle  Davidson...  Pp.  151-170, 

12  figures  in  text.    March,  1918 _ 25 

il.  The  Factors  Controlling  the  Distribution  of  the  Polynoidae  of  the  Pacific 

Coast  of  North  America,  by  Christine  Essenberg.    Pp.  171-238,  plates  6-8, 

2  figures  in  text.    March,  1918--..... _.... „.. .76 

12.  Differentials  in  Behavior  of  the  Two  Generations  of  Salpa  democratica 

Relative  to  the  Temperature  of  the  Sea,  by  Ellis  L.  Michael.    Pp.  239-298, 
plates  9-11, 1  figure  in  text.    March,  1918 —      .65 

13.  A  Quantitative  Analysis  of  the  Molluscan  Fauna  of  San  Francisco  Bay,  by 

E.  L.  Packard.    Pp.  299-336,  plates  12-13,  6  figs.  In  text.    April,  1918 .40 

14.  The  Neuromotor  Apparatus  of  Euplotes  patella,  by  Harry  B.  Yocom.    Pp. 

337-396,  plates  14-16.    September,  1918  „ .70 

15.  The  Significance  of  Skeletal  Variations  in  the  Genus  Feridinium,  by  A.  L. 

Barrows.    Pp.  397-478,  plates  17-20,  19  figures  in  text.    June,  1918 90 


%^:<,rr^  .-_-.,-.^-r-^.^.-:-:^^^ 


UNIVEESITY  OF  CALIFOENIA  PUBLICATIONS— (Continued) 

16.  The  Subclavian  Vein  and  its  Eelations  in  Elasmobranch  Fishes,  by  J. 

Frank  Daniel.    Pp.  479-484,  2  figures  in  text.    August^  1918 .10 

17.  The  Cercaria  of  the  Japanese  Blood  Fluke,  Schistosoma  Japonicum  Kat- 

surada,  by  William  W.  Cort.    Pp.  485-507,  3  figures  in  text. 

18.  Notes  on  the  Eggs  and  Miracidla  of  the  Human  Schistosomes,  by  William 

W.  Cort.    Pp.  509-519,  7  figures  in  text. 

Nos.  17  and  18  in  one  cover.    January,  1919 35 

Index  in  preparation. 

Vol.19.  1.  Eeaction  of  Various  Plankton  Animals  with  Reference  to  their  Diurnal 

Migrations,  by  Calvin  O.  Esterly.    Pp.  1-83.    April,  1919 85 

2.  The  Pteropod  Desmopterus  Pacificiis   (sp.  nov.),  by  Christine  Essenberg. 

Pp.  85-88,  2  figures  in  text „ •. 05 

3.  Studies  on  Giardia  microti,  by  William  C.  Boeck.    Pp.  85-136,  plate  1,  19 

figures  in  text 60 

4.  A  Comparison  of  the  Morphology  and  the  Life  Cycle  of  Crlthidia  with  those 

of  the  Crithidial  Stages  of  Trypanosoma,  by  Irene  A.  McCulloch (In  press) 

5.  A  Mascid  Larva  of  the  San  Francisco  Bay  Region  which  Sucks  the  Blood 

of  Nestling  Birds,  by  O.  E.  Plath.  Pp.  191-200.    February,  1919 10 

Vol.20.  Studies  on  the  Termites,  by  C.  A.  Kofoid  and  Olive  Swezy (In  press) 

Vol.  21.  1.  A  Revision  of  the  Microtus  Calif omicus  Group  of  Meadow  Mice,  by  Rem- 
ington Kellogg.   Pp.  1-42,  1  figure  in  text.    December,  1918 50 

2.  Five  New  Five-toed  Kangaroo  Rats  from  California,  by  Joseph  Grinnell. 

Pp.  43-47.    March,  1919  .05 


NON-GIRGULATING  BOOK 

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